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UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA
Methylglyoxal Metabolism in Leishmania infantum
Lídia Isabel Sebastião Barata
Doutoramento em Bioquímica
(Regulação Bioquímica)
2010
Tese orientada pelo Doutor Carlos Alberto Alves Cordeiro e
pela Doutora Marta Filomena Sousa Silva
DDeeccllaarraaççããoo
De acordo com o disposto no artigo nº. 40 do Regulamento de Estudos Pós-Graduados da
Universidade de Lisboa, Deliberação nº 961/2003, publicada no Diário da República – II Série nº.
153 – 5 de Julho de 2003, foram incluídos nesta dissertação os resultados dos seguintes artigos:
Trincão J‡, Sousa Silva M‡, Barata L, Bonifácio C., Carvalho S., Tomás A. M., Ferreira A. E. N., Cordeiro C.,
Ponces Freire A., Romão M. J. (2006) Purification, Crystallization and Preliminary X-ray Diffraction Analysis
of the Glyoxalase II from Leishmania infantum, Acta Crystallographica Section F 62, 805-807. (‡ both authors
contributed equally for the present work)
Sousa Silva M.‡, Barata L.‡, Ferreira A. E. N., Romão S., Tomás A. M., Ponces Freire A., Cordeiro C.
(2008) Catalysis and Structural Properties of Leishmania infantum Glyoxalase II: Trypanothione
Specificity and Phylogeny, Biochemistry 47, 195-204. (‡ both authors contributed equally for the
present work)
Barata L., Sousa Silva M., Schuldt L., Costa G., Tomás A. M., Ferreira A. E. N., Weiss M. S., Ponces
Freire A., Cordeiro C. (2010) Cloning, expression, purification, crystallization and preliminary X-ray
diffraction analysis of Glyoxalase I from Leishmania infantum, Acta Crystallographica Section F 66,
571–574.
No cumprimento do disposto na referida deliberação, esclarece-se serem da minha
responsabilidade a execução das experiências que estiveram na base dos resultados apresentados
(excepto quando referido em contrário), assim como a interpretação e discussão dos mesmos.
Lisboa, 14 de Julho de 2010
Lídia Isabel Sebastião Barata
Audere est facere.
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AAcckknnoowwlleeddggeemmeennttss// AAggrraaddeecciimmeennttooss
As minhas palavras nunca serão suficientes para agradecer a todos os que, respeitando sempre o que
eu era me tornaram naquilo que sou, e que será inevitavelmente perpetuado no que serei. Naturalmente, esta
tese é o refexo de quatro longos anos de trabalho individual. No entanto, muitas foram as contribuições
directas e indirectas para os bons resultados apresentados, as quais não posso deixar de agradecer. Estando
inserida em dois grupos científicos únicos, e tendo ambos criado um ambiente acolhedor entre muitas
amizades, serão inúmeras as memórias, tanto a nível científico como pessoal, que levarei comigo pela vida.
Em primeiríssimo lugar, gostaria de agradecer aos meus orientadores de doutoramento. À Doutora
Marta Sousa Silva, por ter acompanhado o meu trabalho de muito, muito perto, por ter assistido aos meus
primeiros passos no laboratório, pela sua contribuição especialmente nas primeiras clonagens e purificações
(realizadas na FCUL e no IBMC), pelo incentivo a concorrer a bolsas, congressos e cursos, e sobretudo pela
motivação à minha ida para Hamburgo, por ter tornardo o laboratório cor-de-rosa, e ainda pela amizade que
espero que se perpetue. Ao Doutor Carlos Cordeiro, que supervisionou todo o projecto, pela sua calma,
ponderação e optimismo, por me ter deixado decidir o meu caminho sempre com o seu apoio.
Um muito especial agradecimento à Professora Doutora Ana Ponces Freire, por me ter recebido no
seu grupo, por todo o apoio em situações dificeis, por ser justa, compreensiva e prática, e sobretudo por
representar um exemplo de vida.
Igualmente especial é o meu agradecimento ao Doutor Manfred Weiss... der mich das eine oder
andere Mal in seiner Gruppe empfangen hat, für seine ganze uterstützung, Strenge und Effizienz, sowie für
sein Abschiedsfeier und auch meine.
Não poderia deixar de prestar um enorme reconhecimento a todos os que participaram directamente
neste trabalho. À Professora Ana Tomás (IBMC/ ICBAS), pelo genoma de L. infantum e primers, sem os quais
este projecto não seria possível, e pelo apoio no decurso do trabalho. Do grupo de Enzimologia, FCUL: ao
Gonçalo da Costa, por ter realizado ensaios de espectroscopia de massa que muito contribuiram para este
trabalho, por toda a sua disponibilidade, motivação e companheirismo; e ao Professor António Ferreira pelo
apoio não só informático, como na determinação de parâmetros cinéticos, e pelo insentivo ao uso do
Chimera, o programa de representação de proteínas mais prático que conheço. Do EMBL-HH: meinen dank
an Linda Schuldt, für ihre Hilfe, bei der data collection die ganze Nacht hin durch, der Metallbesrechnung
des Glyoxalase I, aber auch für die stetige Sympathie; an Gerrit Langer, für das Programm der
Metalmengenberechnung des Glyoxalase I; an Xandra Kreplin, für die Kristallisierung versuche,
einschließlich die voll neu Techniken.
Agradeço, ainda, aos restantes elementos e ex-elementos do grupo de Enzimologia da FCUL,
destacando: o Nuno Lages, pelos longos anos de companheirismo, desde os nossos primeiros dias de caloiros
da licenciatura em bioquímica; o Luís Oliveira, por ser sempre amável; o Ricardo Gomes, por criar o bom
humor no laboratório; o Hugo Vicente Miranda, por ter sempre uma ideia para partilhar; a Rita Marques,
pela companhia, pelo mini-gel e pela visita a HH; o Bruno Oliveira, pela sua dor de cotovelo bem
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intencionada que me fazia ter orgulho em mim mesma; o Flávio Figueira, pelo “pedestal”; e a todos eles pela
boa disposição permanente dentro e fora do laboratório.
Do mesmo modo, não posso deixar de agradecer a todas as pessoas que fizeram parte do meu dia-a-
dia em Hamburgo, tornando esta cidade mais quente e única aos meus olhos, e fazendo-me sentir em casa
longe de casa. Meinen dank, an Frank Lehmann, für seine ganze Freundschaft, in sowie auch auβerhalb des
Labors, für die Übergabe des MPD und dafür, dass er mir Gissela vorgestellt hat; an Hubert Mayerhofer für
seine Freundschaft und Sympathie und die Tischfuβballspiele. Merci, a Delphine Chesnel pour aller a la
facilité de cristallisation pour moi, pour les articles que je l’ai demandé de rechercher sous l’internet et pour
tout les drôles moments. Thanks, to Spyros Chatziefthimiou, for being a real gentleman, for the chocolates
and for all the times he said “welcome back”; to Georgios Hatzopoulos for letting me use his desk for a whole
year and for his friendship especially on my visit to Philly; to Elke Noens for her daily enthusiasm; to Chris
Williams, for all the jokes; to Justina Wojdyla and Joannis Manolaridis, for keeping always a contagious good
mood; to Manikandan Karuppasamy, for all the help on my first steps at the EMBL lab, for his kindness and
for the amazing indian cooking; to Matthias Ehebauer, for his extreme calm in a too busy lab; to Chanakya
Nugoor, for his never-ending questions; to Krisztian Fodor, for allowing me to use his thrombin-kit; to
Rositsa Jordanova and Matthew Groves for the help on the SLS data treatment; to Santosh Panjikar, for the
scientific conversations on the way to work; to Saravanan Panneerselvam and Yusuf Akhter for being nice
bench neighbours; to Anne Due, for letting me use her bench for so long; to Philipp Heuser, for all he taught
me about Germany; to Florian Sauer, for the trouble-shootings on the purifiers when no one else was at the
lab. Grazie, a Barbara Tizzano e Viviane Pogenberg, per tentare parlare portoghese; a Francesco Fersini per la
compagnia nella fermata di bus; a Marco Salomone Stagni per la consulenza di uso di filtri ultra-veloce per la
purificazione di tampone, i quali mi hanno salvato molto tempo. Gracias, al Doutor Iñaki de Diego, por la
partilla del peso de un lab vacío en la noche y por su determinación contagiosa; al Daniel Fulla, por la
compañía en las longas noches de beamtime, las inyecciones de autoestima y la “gold”; a Lesley Roca y
Álvaro, por la enormísima simpatía, por las pizzas caseras y todos los buenísimos momentos; a Esther Peña,
por la partilla de la misma situación de ser una “visitante” en HH, por todas las nuestras aventuras
hamburguesas; a Gissela Lehmann, por la hospitalidad, por ser la amiga única que es, por mi maravilloso
cumpleaños 2009, por tener un hijo tan lindo como Vinny, por toda la Inka Kola, los camotes y la calza y por
tenerme enseñado español; a Renzo Perales, que, aunque lejos, de hecho siempre ha estado conmigo en HH,
por ser mí espejo y por la complicidad.
Porque o ambiente lá fora é tão importante como o ambiente dentro do laboratório para que a estrela
da sorte sorria aos bons resultados, não posso deixar de agradecer aos meus amigos que foram rasgando
emoções entre os dias de trabalho.
O maior dos agradecimentos aos meus pais, pelo seu apoio incondicional, por terem confiado sempre
nas minhas capacidades e acreditado sempre no meu sucesso, mesmo quando eu própria não acreditava. Por
terem sido os principais impulsionadores desta minha odisseia pela ciência e por me terem “dado asas”.
Finalmente, agradeço à FCT, MCTES (Portugal) e à EMBO pelo apoio financeiro.
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Table of contents
Acknowledgments/ Agradecimentos ix Summary xiii Resumo xv Abbreviations
xvii
Chapter I – General introduction
1
1. Leishmania and Leishmaniasis 3 1.1. Leishmania infantum life cycle 7 1.2. Trypanosomatids biochemical uniqueness 9
1.2.1. The kinetoplast 9 1.2.2. The glycosome 10 1.2.3. Trypanothione 11
2. Methylglyoxal 13 2.1. Methylglyoxal biosynthesis 14
2.1.1. Methylglyoxal biosynthesis in trypanosomatids 17 2.2. Methylglyoxal catabolism 17
2.2.1. The glyoxalase pathway 18 2.2.1.1. Glyoxalase I 19 2.2.1.2. Glyoxalase II 21 2.2.1.3. The glyoxalase pathway in Trypanosomatids 23
2.2.2. Aldose reductase 25 2.2.2.1. Aldose reductase in Trypanosomatids 26
3. Aims and scope of this work
28
Chapter II – Glyoxalase I from Leishmania infantum
29
1. Summary 31 2. Introduction 32 3. Materials and Methods 34
3.1. Cloning and expression of LiGLO1 34 3.2. Purification of LiGLO1 34 3.3. Metal analysis by Inductively-Coupled Plasma 35 3.4. Crystallization 35 3.5. Data collection and processing 36 3.6. Crystal Structure Solution 37 3.7. Anomalous difference maps 37 3.8. Occupancies of metal binding sites 37 3.9. LiGLO1 Kinetic Analysis 38
4. Results and Discussion 39 4.1. The L. infantum glyoxalase I gene and deduced protein 39
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4.2. Recombinant LiGLO1 over-expressing and purification 39 4.3. Crystals, data collection and processing 40 4.4. Overall description of the LiGLO1 crystal structure 43 4.5. Active Site 45 4.6. Metal composition 46 4.7. LiGLO1 Kinetic Analysis 54 4.8. LiGLO1 evolutive considerations 55
5. Acknowledgements
55
Chapter III – Glyoxalase II from Leishmania infantum
57
1. Summary 59 2. Introduction 60 3. Materials and Methods 62
3.1. LiGLO2 cloning: native and mutant forms 62 3.2. Protein expression and purification 62 3.3. Crystallization 64 3.4. Data collection and crystal structure determination 64 3.5. Metal analysis of recombinant native and mutant glyoxalase II 65 3.6. Enzyme activity assays 65 3.7. Determination of kinetic parameters 66 3.8. Activity in crystals 66
3.9. Evolutionary analysis 66 4. Results 67
4.1. The L. infantum glyoxalase II gene and deduced protein 67 4.2. Over-expression and purification of recombinant glyoxalase II 71 4.3. Final structures 71 4.4. The active site 76 4.5. The substrate-binding site 77 4.6. LiGLO2 kinetics: specificity for thiolesters of SPD-GSH conjugates 78 4.7. Mutant form of LiGLO2 78 4.8. Trypanothione specificity: structural and evolutionary analysis 82
5. Discussion 83 6. Acknowledgements
86
Chapter IV – Aldose Reductase from Leishmania infantum
87
1. Summary 89 2. Introduction 90 3. Materials and Methods 91
3.1. Cloning of LiAKR 91 3.2. Expression of LiAKR in different E. coli strains and protein solubility 91 3.3. Purification buffer optimization by ThermoFluor 92 3.4. Large scale production and purification from soluble fraction 92 3.5. Protein analysis by mass spectrometry 93
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3.6. Recombinant LiAKR activity assay 93 4. Results and Discussion 93
4.1. Cloning, expression and purification of LiAKR 94 4.2. Purification buffer optimization by ThermoFluor 96 4.3. Protein analysis by mass spectrometry 97 4.4. Activity of recombinant LiAKR 97
5. Concluding remarks 98 6. Acknowledgements
99
Chapter V – Concluding remarks
101
References 109
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SSuummmmaarryy
Leishmaniasis, caused by a Leishmania parasite belonging to the Trypanosomatidae family,
are diseases affecting humans and other mammals. The most severe form of the disease is lethal if
untreated, currently existing no vaccines or efficient therapies. The identification of new therapeutic
targets is presently based on exploiting the biochemical differences between the parasite and the
host. One of the main biochemical characteristics distinguishing trypanosomatids from other
eukaryotic cells is the functional replacement of glutathione by trypanothione. In trypanosomatids,
the glyoxalase system, comprising the enzymes glyoxalase I and glyoxalase II, depends on
trypanothione to eliminate methylglyoxal, a toxic compound formed non-enzymatically during
glycolysis. Hence, this is an excellent model system to understand trypanothione-dependent
enzymes specificity at a kinetic and molecular level. The methylglyoxal metabolism in L. infantum
study would not be complete without an account of aldose reductase, a NADPH-dependent
enzyme also catabolising this toxic compound. This project includes an eclectic structural and
biochemical study of the main enzymes involved in methylglyoxal catabolism, contributing for the
knowledge of these complex parasites. Additionally, these enzymes’ activities complement each
other in such a way that they can be synergistically exploited in the quest for new anti-leishmanial
drug targets.
The glyoxalase I gene from Leishmania infantum (LiGLO1) was isolated and cloned into an
expression vector for bacteria. The recombinant protein was over-expressed in E. coli, purified and
kinetically characterised. LiGLO1 showed to preferentially use the hemithioacetal derived from
trypanothione, although it can also catalyse the same reaction with the glutathione-derived
hemithioacetal. The recombinant protein was crystallised and its structure solved by molecular
replacement, using the glyoxalase structure from L. major as a search model. Although the LiGLO1
structure is very similar to the L. major GLO1, as expected by its high homology, the metal observed
at the active site is different. While LmGLO1 requires nickel for its activity, like glyoxalase I from
prokaryotes, it was shown both by ICP and anomalous diffraction that LiGLO1 contains zinc in the
active site, as its eukaryotic homologues. On the other hand, LiGLO1 has significant structural
differences relatively to the human glyoxalase I enzyme.
The glyoxalase II gene from L. infantum (LiGLO2) was also isolated and cloned in a bacterial
expression vector. The recombinant protein was over-expressed in E. coli, purified and kinetically
characterised, confirming its specificity towards trypanothione-derived thiolesters. LiGLO2 was
crystallised and its structure solved by molecular replacement, using the glutathione-dependent
SSuummmmaarryy
xxiivv
human glyoxalase II structure as a search model, for its high sequence homology with the
structurally unknown L. infantum protein. The determined structural model for LiGLO2 is very
similar to its human counterpart. Highly conserved residues were identified in the active site, as
well as specific residues of the L. infantum enzyme, being noteworthy the presence of the
spermidine-binding Cys294 and Ile171, both absent from the human enzyme. The presence of a
spermidine molecule on the LiGLO2 substrate binding site, together with sequence analysis,
clarified the enzyme’s substrate specificity at a molecular level. Both ICP metal-analysis and the B
factor values for the metal atoms revealed the presence of zinc and/or iron in the enzyme active
site. A structure with D-lactate in the active site was obtained by crystal soaking with substrate.
Superimposing both LiGLO2 structures, the localization of the trypanothione-derived thiolester in
the substrate-binding site could be clearly inferred. Two of the residues forming the substrate-
binding pocket, Tyr291 and Cys294, were subsequently replaced by the S-D-lactoylglutathione-
binding residues found on the human enzyme, Arg249 and Lys252, respectively. Recombinant
mutated LiGLO2 was over-expressed in E. coli. Kinetic analysis revealed that the enzyme’s
substrate specificity was changed, catalysing the reaction with S-D-lactoylglutathione, and loosing
affinity towards S-D-lactoyltrypanothione. These results show that the mutated residues are critical
for the enzyme specificity.
The aldose reductase gene from L. infantum (LiAKR) was identified for the first time in a
trypanosomatid. It was isolated and cloned into an expression vector. Over-expression of the
soluble recombinant protein in E. coli was only achieved by co-expression with chaperone systems.
The LiAKR enzyme was kinetically characterised as a NADPH-dependent aldose reductase
involved in the catabolism of methylglyoxal. This protein was recently crystallised, although the
observed diffraction requires crystal optimization.
Keywords: Leishmania infantum, methylglyoxal, trypanothione, glyoxalase pathway, aldose
reductase.
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RReessuummoo
Leishmanioses são doenças que afectam humanos e outros mamíferos, provocadas por um
parasita do género Leishmania, pertencente à família Trypanosomatidae. A forma mais severa da
doença é letal se não for tratada, não existindo actualmente vacinas ou terapias curativas eficazes. A
identificação de novos alvos terapêuticos baseia-se em diferenças encontradas entre o parasita e o
hospedeiro. Uma das principais características bioquímicas que distingue os tripanossomatídeos de
outras células eucariotas é a substituição funcional de glutationo por tripanotiono. Em
tripanossomatídeos, o sistema dos glioxalases, constituído pelos enzimas glioxalase I e glioxalase II,
depende de tripanotiono para eliminar o metilglioxal, um composto tóxico formado não-
enzimaticamente durante a glicólise. Assim, este é um excelente sistema modelo para compreender a
especificidade de enzimas dependentes de tripanotiono a um nível cinético e molecular. No entanto,
o estudo do metabolismo do metilglioxal em L. infantum não estaria completo sem referir o aldose
redutase, um enzima dependente de NADPH que também catabolisa este composto tóxico. Este
projecto inclui um estudo estrutural e bioquímico eclético dos principais enzimas envolvidos no
catabolismo do metilglioxal, contribuindo para o estudo destes complexos parasitas. Para além
disso, as actividades destes enzimas são de tal modo complementares que podem ser exploradas
sinergisticamente na busca por novos alvos para fármacos anti-leishmania.
O gene do glioxalase I de Leishmania infantum foi isolado e clonado num vector de expressão. A
proteína recombinante foi sobre-expressa em E. coli, purificada e caracterizada cineticamente, verificando-se
que o LiGLO1 catalisa preferencialmente o hemitioacetal derivado de tripanotiono, embora também possa
utilizar o hemitioacetal derivado de glutationo. A proteína recombinante foi cristalizada e a sua estrutura
resolvida por substituição molecular, utilizando a estrutura do glioxalase I de L. major como modelo de
pesquisa. Embora a estrutura do LiGLO1 seja muito semelhante à do LmGLO1, como esperado pela sua
elevada homologia de 97 %, estes enzimas diveregem no metal presente no centro activo. Enquanto o
LmGLO1 requer níquel para a sua actividade, à semelhança dos glioxalases I de procariotas, foi verificado
tanto por ICP como por difracção anómala, que o LiGLO1 contém zinco no seu centro activo, à semelhança
dos seus homólogos eucariotas. Estes resultados permitiram-nos estabelecer uma diferença entre ambas as
espécies de Leishmania, e propor uma hipótese em que o enzima de L. infantum terá divergido dos procariotas
mais cedo na linha evolutiva do que o enzima de L. major. Por outro lado, LiGLO1 tem grandes diferenças
estruturais em relação ao homólogo humano.
O glioxalase II de L. infantum foi também isolado e clonado num vector de expressão. A proteína
recombinante foi sobre-expressa em E. coli, purificada e caracterizada cineticamente, confirmando-se a sua
especificidade para os tioésteres derivados de tripanotiono. LiGLO2 foi cristalizado e a sua estrutura resolvida
RReessuummoo
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por substituição molecular, utilizando a estrutura do glioxalase II humano, dependente de glutationo, como
modelo de pesquisa. O modelo da estrutura determinado para o LiGLO2 é muito semelhante ao do seu
homólogo humano. Foram identificados resíduos conservados no centro activo (His76, His78, Asp80, His81,
His139, Asp164, His210) bem como resíduos específicos no enzima de L. infantum (dos quais é de salientar a
presença da Cys294 e Ile171, que ligam a espermidina e a ausência dos resíduos Arg249, Lys143 e Lys252,
existentes no enzima humano). A presença de uma molécula de espermidina no local de ligação ao substrato
do LiGLO2, juntamente com uma análise das sequências, elucidou a especificidade do substrato do enzima ao
nível molecular. Tanto a análise de metais por ICP, como os valores dos factores B dos átomos de metal,
revelaram a presença de zinco e/ou ferro no centro activo do enzima. Foi obtido um modelo estrutural deste
enzima com D-lactato no centro activo, através de soaking de cristais com substrato. Sobrepondo ambas as
estruturas do LiGLO2, pôde ser claramente inferida a localização do tioester derivado de tripanotiono no
centro de ligação ao substrato. Dois dos resíduos que formam a cavidade onde se liga o substrato, Tyr291 e
Cys294, foram subsequentemente substituídos por resíduos que ligam o S-D-lactoilglutationo no enzima
humano, Arg249 e Lys252, respectivamente. O LiGLO2 mutado recombiante foi sobre-expresso em E. coli.
Este enzima mutado foi instável durante o processo de purificação. Análise cinética revelou que a
especificidade do enzima para o substrato foi alterada, reagindo com S-D-lactoilglutationo e perdendo
actividade para o S-D-lactoiltripanotiono. Estes resultados mostram que os resíduos mutados são críticos para
a especificidade do enzima.
O gene do aldose redutase de L. infantum foi identificado pela primeira vez num tripanossomatídeo.
Foi isolado e clonado num vector de expressão. A sobre-expressão da proteína recombinante na fracção
solúvel em E. coli foi apenas conseguida por co-expressão com sistemas de chaperones. O enzima LiAKR foi
cineticamente caracterizado como um aldose redutase dependente de NADPH envolvido no catabolism do
metilglioxal. Esta proteína foi recentemente cristalizada, embora a difracção observada requeira optimização
dos cristais.
Palavras-chave: Leishmania infantum, metilglioxal, tripanotiono, via dos glioxalases, aldose
redutase.
AAbbbbrreevviiaattiioonnss
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AAbbbbrreevviiaattiioonnss Å Angström
A; Abs Absorbance
Acetyl-CoA Acetyl-coenzyme A
ADP Adenosine 5’-diphosphate
AGE Advanced glycation end-product
AIDS Acquired immune deficiency syndrome
AKR Aldo-keto reductase
AKR1A Human aldehyde reductase; aldehyde:NAD(P)+ oxidoreductase; EC 1.1.1.2
AKR1B Human aldose reductase; alditol:NAD(P)+ oxidoreductase; EC 1.1.1.21
ATP Adenosine 5’-triphosphate
Bj Thermal vibration factor
1,3-BPGA 1,3-biphosphoglycerate
BSA Bovine serum albumin
Co-NTA Cobalt-nitrilotriacetic acid
CoA Coenzyme A
dmin Maximum resolution of a diffraction pattern
Da Dalton
DESY Deutsches Elektronen-Synchrotron
DHAP Dihydroxyacetone phosphate
DNA Deoxyrribonucleic acid
DTT Dithiothreitol
Ε Molar absorptivity coefficient
EDTA Ethylene diamine tetraacetic acid
EGTA Ethylene glycol tetraacetic acid
ESRF European Synchrotron Radiation Facility
eV Electron volt
FPLC Fast protein liquid chromatography
Fructose-1,6-BP D-Fructose-1,6-bisphosphate
GAP D-Glyceraldehyde-3-phosphate
GAPDH D-Glyceraldehyde-3-phosphate dehydrogenase: NADP+ oxidoreductase;
EC 1.2.1.9
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Glycerol-3-P D-Glycerol-3-phosphate
6-P-Glucose D-Glucose-6-phosphate
GPDH D-Glycerol-3-phosphate dehydrogenase;
sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase; EC 1.1.1.8
GSH Glutathione, γ-glutamilcisteynilglicine
GspdSH N1-Glutathionylspermidine
GLO1 Glyoxalase I; lactoylglutathione lyase; EC 4.4.1.5
GLO2 Glyoxalase II; hydroxyacylglutathione hydrolase, EC 3.2.1.6
GLO4 Glyoxalase II mitochondrial isoform;
hydroxyacylglutathione hydrolase, EC 3.2.1.6
GRE3 Yeast aldose reductase gene, Gene de Respuesta al Estress
GSSG Oxidised glutathione
Hepes N-(2-hydroxyethyl)piperazine-2-ethanesulfonic acid
HIV Human immunodeficiency virus
HTA Hemithioacetal
ICP Inductively coupled plasma
IPTG Isopropil-β-D-thiogalactopyranoside
kDNA Kinetoplast DNA
kcat Catalytic constant
Km Michaelis-Menten constant
LB Luria-Bertani
LiGLO1 Glyoxalase I from Leishmania infantum; lactoylglutathione lyase; EC 4.4.1.5
LiGLO2 Glyoxalase II from Leishmania infantum; hydroxyacylglutathione hydrolase;
EC 3.2.1.6
LiAKR Aldose reductase from Leishmania infantum; alditol:NAD(P)+ oxidoreductase;
EC 1.1.1.21
M Molar
MAGE Methylglyoxal-derived advanced glycation
MALDI-FTICR-MS Matrix assisted laser desorption ionization –
Fourier transform ion cyclotron resonance mass spectrometry
MALDI-TOF-MS Matrix assisted laser desorption ionization –
time of flight mass spectrometry
MES 2-n-morpholino-ethanesulfonic acid
MG Methylglyoxal
AAbbbbrreevviiaattiioonnss
xxiixx
MLT Mono-S-(lactoyl)-trypanothione
MOPS 3-(N-Morpholino)propanesulfonic acid
MOLD Methylglyoxal-lysine dimer
MPD 2-Methyl-2,4-pentanediol
Mr Molecular weight
MR Molecular replacement
mRNA Messenger RNA
NAD+ Nicotinamide adenine dinucleotide, oxidised form
NADH Nicotinamide adenine dinucleotide, reduced form
NADP+ Nicotinamide adenine dinucleotide phosphate, oxidised form
NADPH Nicotinamide adenine dinucleotide phosphate, reduced form
Ni-NTA Nickel-nitrilotriacetic acid
NMWL Nominal molecular weight limit
NTA Nitrilotriacetic acid
OD Optical density
PBS Saline phosphate buffered
PCR Polymerase chain reaction
PEG Poliethylene glycol
PFK 6-Phosphofructokinase; ATP: D-fructose-6-phosphate 1-phosphotransferase;
EC 2.7.1.11
3-PGA 3-Phosphoglycerate
PMF Peptide mass fingerprint
Rmsd Root mean square deviation
RNA Ribonucleic acid
RNAi RNA interference
Rpm Rotations per minute
σ (I) Error associated to intensity
S Soluble fraction
SSAO Semicarbazide-sensitive amine oxidase; EC 1.4.3.6
SDL-GSH S-D-lactoylglutathione
SDL-GspSH S-D-lactoylglutathionylspermidine
SDL-T(SH)2 S-D-lactoyltrypanothione
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
AAbbbbrreevviiaattiioonnss
xxxx
Spd Spermidine; N-(3-aminopropyl)butane-1,4-diamine
T Total cell content
TFK Potassium phosphate buffer
TIM Triosephosphate isomerase; D-glycer- aldehyde-3-phosphate ketol-isomerase,
EC 5.3.1.1
Tm Temperature midpoint of the protein-unfolding transition
TS2 Oxidised trypanothione; oxidised N1,N8-bis(glutathionyl)-spermidine
TR Trypanothione reductase; EC 1.6.4.8
Tris Trishydroxymethylaminomethane
T(SH)2 Reduced trypanothione; reduced N1,N8-bis(glutathionyl)-spermidine
U Enzymatic activity units
V Limiting rate
VM Mathews coefficient
% v/v Percentage expressed in volume/volume
% w/v Percentage expressed in weight/volume
WHO World Health Organization
CChhaapptteerr II GGeenneerraall IInnttrroodduuccttiioonn
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11.. LLeeiisshhmmaanniiaa aanndd LLeeiisshhmmaanniiaassiiss
Leishmaniasis is a group of diseases caused by flagellated protozoa from the
Trypanosomatidae family. Belonging to the Kinetoplastida Order, this family integrates several
protozoan parasites, affecting humans, animals, plants and insects. Besides Leishmaniasis, some of
the human diseases caused by these etiologic agents are the sleeping sickness and Chagas’ disease,
caused by the parasites Trypanosoma brucei and Trypanosoma cruzi, respectively (for review see
Castro & Tomás 2008). These parasites are also responsible for animal diseases, as reported for T.
Brucei brucei, T. congolense and T. vivax causing Nagana in cattle in Africa, and Leishmania species
leading to leishmaniasis in a range of animals from rodents (Shaw & Lainson 1968), to guinea-pig
(Bryceson et al. 1970) and dogs (Ciaramella et al. 1997).
Leishmania is one of the nine genera of the Trypanosomatidae, a classification based on their
morphological features. The other genera are: Trypanosoma, Blastocrithidia, Crithidia, Endotrypanum,
Herpetomonar, Leptomonas, Phytomonas and Sauroleishmania (Hoare 1966 as in Hoare 1967). Although
phylogenetically close, trypanosomatids share many biochemical traits. Mutual exclusivity can arise
even between two Leishmania species, making them surprisingly unique, as will be discussed along
this work.
Leishmania is transmitted by a female sandfly. The parasite’s name, and consequently the
disease, comes from William Leishman who in 1901 identified the organisms in the spleen of a
patient who had died from “dum-dum fever”. This disease was
characterised by general debility, irregular fever, severe
anaemia, muscular atrophy and excessive swelling of the spleen
(Leishmaniasis. A brief history of the disease, WHO 2010).
Another form of the disease, cutaneous leishmaniasis, seems to
have existed way back in history, as proved by representations
of skin lesions and facial deformities, evidence of cutaneous and
mucocutaneous forms of leishmaniasis, found on first century
pre-Inca potteries from Ecuador and Peru (Figure I.1.)
(Leishmaniasis. A brief history of the disease, WHO 2010). The
disease was often registered along history, as illustrated by the
15th and 16th centuries Inca texts mentioning the "valley
sickness" or "Andean sickness", as consisting on skin lesions,
Figure I.1. – Pre-Inca pottery, “Huaco Mochica”, showing leishmaniasis lesions in the nose and upper lip (as first suggested by Ashmead 1900; picture as in Altamirano-Enciso et al. 2003).
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common to the Andes seasonal agricultural workers. Later, the disease was also referred to as
"white leprosy". On the other hand, the presently defined visceral leishmaniasis, was described in
India by physicians who named it kala-azar (meaning "black fever") (Leishmaniasis. A brief history
of the disease, WHO 2010).
It is estimated that twelve million people in the World are affected by one of the diverse
forms leishmaniasis (cutaneous, visceral or muco-cutaneous). About two million new cases (500,000
of visceral leishmaniasis) appear every year in eighty-eight countries spread through Mediterranean
Europe, South America, Central Asia and Africa (data from Leishmaniasis. Burden of disease, WHO
2010). It is estimated that more than 350 million people are at risk (Leishmaniasis. Initiative for
Vaccine Research, WHO 2010). The organisms observed by Leishman, were only separated from
trypanosomes in 1903, by Captain Donovan (Donovan 1903 in Bailey & Bishop 1959). In the same
year, Major Ross associated these organisms to the kala-azar disease, naming them Leishmania
donovani and creating the Leishmania genus (Ross 1903 in Bailey & Bishop 1959). Altogether, there
are about twenty Leishmania species pathogenic for humans that are transmitted by thirty of the five
hundred known sandfly species (Figure I.2.) (Leishmaniasis. The disease and its epidemiology,
WHO 2010). According to the involved vector and the affected area of the World, Leishmania
parasites can be considered as New World or Old World (Figure
I.3.). In the first group, parasites are transported by a vector of
the Lutzomyia genus which, being a permissive vector, transports
a range of Leishmania species, including L. chagasi, L. braziliensis
or L. amazonensis (Sharma & Sarman Singh 2008). Old World
Leishmania are transported by a vector of the genus Phlebotomus.
Both Phlebotomus papatasi and P. sergenti are specific for L. major
and L. tropica, respectively. Although Phlebotomus argentipes is
more tolerant to L. donovani and L. infantum, it can also be the
vector for L. major, L. tropica and L. amazonensis (Sharma &
Sarman Singh 2008).
Figure I.2. Phlebotomine sandfly (Stanford University, USA, available from: www.stanford.edu/class/humbio103/ParaSites2003/Leishmania)
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Figure I.3. World distribution of visceral leishmaniasis (WHO 2003, available from:
www.who.int/leishmaniasis/leishmaniasis_maps/en/index.html). Incidence of leishmaniasis is shown to
affect eighty-eight countries, both in New World and Old World. A characteristic sandfly genus is responsible
for transportation of the parasite: Lutzomyia in the New World, and Phlebotomus in the Old World.
Leishmaniasis forms differ in severity and manifestations, and can be either zoonotic, as
when caused by L. infantum (Tesh 1995), or anthroponotic disease, as L. donovani (Rosypal et al.
2010). The different forms of leishmaniasis can be divided in two major groups: cutaneous
leishmaniasis and visceral leishmaniasis. In cutaneous leishmaniasis (also known as oriental sore,
Delhi ulcer, Aleppo, Delhi or Baghdad boil) the parasite replicates within macrophages located in
the dermis and the disease is manifested as skin lesions. In visceral leishmaniasis (also known as
kala-azar, black disease, dum-dum fever) the parasite replicates within the bone marrow, spleen or
liver and the disease is associated with fever, hepatosplenomegaly, anaemia and other life
threatening symptoms (Murray et al. 2005). The type of disease depends on the parasite species:
L. tropica (L. t. major, L. t. minor and L. ethiopica) and L. mexicana cause the cutaneous form of the
disease (Figure I.4a.), while L. donovani, L. infantum and L. chagasi lead to the visceral leishmaniasis
form (Figure I.4b.; Sharma & Sarman Singh 2008).
The third form of the disease, mucocutaneous leishmaniasis (espundia, Uta, Chiclero ulcer)
(Leishmaniasis. The disease and its epidemiology, WHO 2010; Sharma & Sarman Singh 2008), is
caused by, for example, L. (viannia) braziliensis and L. (viannia) guyanensis. In the case, the parasite
replicates within macrophages located in the naso-oropharyngeal mucosa (Sharma & Sarman Singh
2008).
(a) (b) (c)
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We can also consider another less common form of the disease, the diffuse cutaneous or
mucocutaneous leishmaniasis, caused primarily by L. mexicana, L. ethiopica and L. donovani
(Leishmaniasis. The disease and its epidemiology, WHO 2010; Sharma & Sarman Singh 2008),
characterised by non-ulcerating nodules over the entire body (Figure I.4c.). This leishmaniasis form
commonly appears as a relapse manifested by skin lesions, after leishmania symptoms attenuation
(especially when in the visceral form). In this case, the syndrome is called kala-azar dermal
leishmaniasis and attributes to the patient a reservoir host role (Stark et al. 2006).
Figure I.4. Forms of leishmaniasis. (a) Child showing a cutaneous leishmaniasis lesion on the face (The
Welcome Trust 2000, Leishmaniasis, Topics in International Health). (b) Profile view of a child suffering from
visceral leishmaniasis, exhibiting splenomegaly, distended abdomen and severe muscle wasting (The
Welcome Trust 2000, Leishmaniasis, Topics in International Health). (c) Girl with diffuse mucocutaneous
leishmaniasis of the face, responding to treatment (WHO/TDR/El-Hassan).
Leishmaniasis gain dramatic relevance in areas where occurs co-infection with HIV
(Montalban et al. 1990). This is due to the development of atypical symptoms by individuals with
immune system deficiencies, apart from their higher susceptibility to the disease (Angarano et al.
1998). Although often neglected, in 1990, visceral leishmaniasis was estimated to be one of the most
frequent parasitic disease co-infecting AIDS patients, in Spain (Montalban et al. 1990).
Leishmaniasis can be lethal, especially when in the visceral form. There is currently no
vaccine against these diseases. Both visceral and cutaneous leishmaniasis are traditionally treated
with pentavalent antimonials, which are toxic and decreasing their efficiency due to emerging
trypanosomatid resistance (Sundar 2001). The alternative drugs, such as Amphotericin B,
Pentamine and Miltefosine have low efficiencies and high costs. These drawbacks, together with the
emergence of resistant trypanosomatids, demand the investigation of these parasites’ metabolism
targeting new therapeutic approaches.
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11..11.. LLeeiisshhmmaanniiaa iinnffaannttuumm lliiffee ccyyccllee
L. infantum life cycle alternates between phagolysosomes of the vertebrate host (e.g. human)
macrophages and the alimentary tract of an insect vector (sandfly) (Figure I.5.). Only the female
sandfly transmits the parasite, as it requires blood to obtain the required proteins for the eggs
development (Leishmaniasis. The disease and its epidemiology, WHO 2010). In the sandfly, the
parasite grows for four to twenty-five days as flagellated extracellular promastigotes
(Leishmaniasis. The disease and its epidemiology, WHO 2010), asexually reproducing (by binary
fission) in the sandfly midgut (Hepburn et al. 2003). Requiring organic matter, heat and humidity to
the larvae survival, the female sandfly lays its eggs in places as the bark of old trees, ruined
buildings, animal shelters or household rubbish (Sharma & Singh 2008).
Figure I.5. Leishmania spp. life cycle. Promastigotes reproduce in the vector midgut. When injected into the
vertebrate host, they are phagocytised by macrophages, where they shift their morphologic form to
amastigotes. At the macrophages’ death, amastigotes are released to infect other cells (Kamhawi et al., 2004).
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Figure I.6. L. infantum parasite forms. (a) Promastigote (amplification 1000x; picture taken by R. L. Jacobson 1996). (b) Several amastigotes in a macrophage cytoplasm (Joiner et al. 2005).
Promastigotes (Figure I.6.) are introduced into
the host during a blood meal taken by the sandfly,
usually in the evening and in a radius of several
hundred meters from its habitat (Sharma & Singh
2008). The parasites are subsequently phagocytosed by
macrophages in the skin, liver, spleen and bone
marrow and suffer a metamorphosis, where they
change their morphological form to intracellular
amastigotes (Herwaldt et al. 1999). Amastigotes
(Figure I.6.), a parasite form without flagella and smaller than promastigotes, reproduce in the
macrophages, being freed at their death to infect other cells (Kamhawi et al., 2004).
Studies on Leishmania differentiation, comparing promastigote and amastigote gene and
protein expression, revealed that both parasite forms have reached a high level of adaptation to
their very distinct environments (Rosenzweig et al. 2008). Respiration, catabolism of energy
substrates and synthesis of macromolecules are explicit example processes of the latter adaptation.
In amastigotes, these metabolic processes occur in an acidic pH, while in promastigotes the optimal
pH is neutral (Rosenzweig et al. 2008), similar to the parasite´s respective environmental pH
(Opperdoes & Coombs 2007). Another striking aspect is the different activation extent of
fundamental pathways: glycolysis, more active in promastigotes; the fatty acid oxidation, more
active in amastigotes (Opperdoes & Coombs 2007, Rosenzweig et al. 2008); or gluconeogenesis,
essential for the amastigotes infectiveness and proliferation in the macrophages and not relevant in
promastigotes (Naderer et al. 2006, Rosenzweig et al. 2008). On the other hand, the identification of
stage-specific genes supports the adjustment of each of the
Leishmania forms to its environment at a genetic level
(Zhang et al. 1996; Bates 1993; Wiese 1998; Bente et al. 2003;
Akopyants et al. 2004; Almeida et al. 2004; Nugent et al. 2004;
Walker 2006; Huynh et al. 2006; Rosenzweig et al. 2008).
Although different characteristics for promastigotes
and amastigotes are presently known, as mentioned, the
molecular mechanisms underlying the reason for and the
morphological change are not completely understood.
However, differentiation in L. donovani, the most similar
Figure I.7. Promastigotes in aggregation stage (8h). Amplification 40x. Picture courtesy of Renzo Perales.
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Leishmania species to L. infantum, was revealed to be a regulated process as far as it is accompanied
by changes in gene and protein expression (Saar et al. 1998; Barak et al. 2005; Saxena et al. 2007;
Rosenzweig et al. 2008). It was inferred that differentiation had four main stages (as described in
Rosenzweig et al. 2008): i) 0–4 h, differentiation signal is received by promastigotes; ii) 5–9 h,
promastigotes slow down their motion and aggregate (Figure I.7.); iii) 10–24 h, promastigotes
undergo morphological change into amastigotes; and iv) 25–120 h, amastigotes maturation. At the
beginning of the differentiation process almost all gene transcripts are down-regulated, while
transcripts specific for amastigotes are up-regulated in the fourth stage described (Rosenzweig et al.
2008). However, changes in mRNA do not to dependably affect proteins amounts. Regulation of
protein activity seems to occur mainly by posttranscriptional modifications (Clayton et al. 2002).
Furthermore, Besteiro and co-workers investigated the protein remodelling by autophagy as an
essential process for promastigote-to-amastigote differentiation (Besteiro et al. 2006, Rosenzweig et
al. 2008).
11..22.. TTrryyppaannoossoommaattiiddss BBiioollooggiiccaall UUnniiqquueenneessss
Being invasive parasites of host cells, trypanosomatids need to be adapted not only to both
extra and intracellular environments, but also to the abrupt change they undergo between the two
very different and extreme conditions. Consequently, these organisms developed unique
characteristics along evolution, some of the most relevant being the kinetoplast, the glycosome and
the thiol trypanothione. These trypanosomatid-specific features inevitably attract the
parasitologists’ attention towards the identification of new therapeutic targets.
1.2.1. The Kinetoplast
The Kinetoplast, a mitochondrial organelle, located near the basal body, encloses the
network formed by the two forms of kDNA (kinetoplast DNA): the maxicircle and the minicircle.
The former includes large molecules at low copy number and correspond to the conventional
mitochondrial DNA, while the latter comprises small molecules in high copy number, functionally
related to the editing process of maxicircle kDNA (Simpson 1987; Sturm and Simpson 1990,
Brandão 2000). Kinetoplast features in general, and minicircles in particular, are used for
trypanosomatid diagnosis (Morel et al. 1980; Sturm et al. 1989; Degrave et al. 1994; Fernandes et al.
1996).
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1.2.2. The Glycosome
Another important trypanosomatids characteristic is the unique sequestering of glycolysis
and other pathways of carbohydrate metabolism in specialised organelles, designated glycosomes
(Opperdoes & Borst, 1977). Glycosomes are similar to peroxisomes for having an outer
phospholipidic layer, not enclosing DNA and including a matrix of proteins which are synthesised
in the cytosol and then imported. However, unlike the peroxisomes, the glycosomes are essential
organelles (Furuya et al. 2002).
Figure I.8. Glycolysis and hypothetical glyoxalase pathway location in Leishmania. Solid lines represent
reactions catalysed by a single enzyme; dashed lines represent multiple sequential reactions. Fructose-1,6-BP,
fructose-1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; 1,3-BPGA, 1,3-biphosphoglycerate; 3-PGA, 3-
phosphoglycerate; DHAP, dihydroxyacetone phosphate; Glycerol-3-P, glycerol 3-phosphate; Pi, inorganic
phosphate; GLO1, glyoxalase I; GLO2, glyoxalase II. Adapted from (Opperdoes & Coombs 2007).
The vital importance of the processes occuring in the glycosome has led to the notion that
this metabolic compartmentation itself might be essential as well. In 1997, Bakker and co-workers
discovered that the glycolytic flux could only be controlled if a part of the pathway was considered
to occur inside the glycosome (Bakker et al. 1997, Myler & Fasel 2008). Enclosed by the glycosomal
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membrane, impermeable to metabolites and co-enzymes (Visser et al. 1981; Opperdoes, 1987;
Clayton and Michels, 1996; Blattner et al. 1998), are the first seven of the nine glycolytic enzymes
which convert D-glucose to 3-phosphoglycerate, being present in the cytoplasm only the last three
enzymes of this pathway (Opperdoes and Borst, 1977; Opperdoes, 1987; Hannaert and Michels,
1994; Hannaert et al. 2003; Moyersoen et al. 2004) (Figure I.8.). The glycosome and the enclosed
metabolic processes, including or not the methylglyoxal catabolism (Figure I.8.), are nowadays
considered relevant candidates for drug targets (Myler & Fasel 2008).
1.2.3. Trypanothione
Efficient antioxidant systems are an adaptation to the hostile environment faced by
trypanosomatids when invading the host, where oxidants as peroxynitrite, hypochlorite, and H2O2
are a constant threat (Jager & Flohé 2006). These systems include around twenty thiol-dependent
proteins (Krauth-Siegel et al. 2005) and the unique thiol trypanothione (T(SH)2, N1,N8-
bis(glutathionyl)spermidine) (Fairlamb et al. 1985), a conjugate of two molecules of glutathione
(GSH) with the polyamine spermidine.
Functionally, trypanothione replaces glutathione in trypanosomatids, being the glutathione-
dependent enzymes replaced by functionally analogue enzymes using trypanothione (Muller et al.
2003). Like GSH, T(SH)2 is reduced from its oxidised form by a NADPH-dependent trypanothione
reductase (EC 1.6.4.8, TR; Shames et al. 1986). Producing trypanothione, these organisms’ specific
oxidation-reduction mechanism is based in the trypanothione/ trypanothione reductase conjugate
(Fairlamb et al. 1985), replacing the pair glutathione/ glutathione reductase (Schmidt and Krauth-
Siegel 2003).
In trypanosomatids, as in other organisms, glutathione is synthesised by consecutive activity
of the enzymes γ-glutamylcysteine synthetase (EC 6.3.2.2; Hibi et al. 2004) and glutathione
synthetase (EC 6.3.2.3; Fyfe et al. 2010). For spermidine, however, the biosynthetic pathway diverges
according to the trypanosomatid species (Bacchi et al. 2007).
T(SH)2 is synthesised by the conjugation of spermidine with two glutathione molecules
(Figure I.9.) (Flohe et al. 1999; Oza et al. 2005). Its synthesis begins with N1 or N8-monoglutathionyl-
spermidine formation, catalysed by the glutathionyl-spermidine sinthetase (γ-L-glutamil-L-
cysteinil-glycine: spermidine ligase, EC 6.3.1.8). Trypanothione synthetase (glutathionyl-
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spermidine: glutathione ligase, EC 6.3.1.9) catalyses the monoglutathionyl-spermidine ligation to a
second GSH molecule, forming T(SH)2. Trypanothione formation can be inhibited at the
glutathionyl-spermidine level, by compounds similar to spermidine or glutathione (Henderson et al.
1990; Verbruggen et al. 1996). In L. major, trypanothione synthetase can catalyse both T(SH)2
formation steps (Oza et al. 2005).
Figure I.9. Synthesis reactions of glutathionyl-spermidine and trypanothione, from glutathione and
spermidine (Oza et al. 2002).
As a reducing agent, T(SH)2 shows an oxidation-reduction potential of -242 mV, similar to
GSH (-230 mV). However, being a dithiol, T(SH)2 has two reducing disulfide groups available for
reaction, which is equivalent to two GSH molecules. Hence, it is kinetically more favourable as a
reductant than GSH (Gilbert 1990; Krauth-Siegel et al. 2005). On the other hand, these thiols differ in
their pKa value. Compared to the GSH pKa value of 8.7-9.2, T(SH)2 has a lower pKa (7.4) (Moutiez et
al. 1994), probably due to the nitrogen atom positively charged in the spermidine bridge (Krauth-
Siegel et al. 2005). Moreover, high T(SH)2 reactivity, when compared to GSH, arises in part from its
pKa value overlapping the pH in the trypanosomatids environment (Fraser-L’Hostis et al. 1997), as
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the velocity second-order constants are optimal when the thiol pKa matches the media solution pH
(Gilbert 1990; Krauth-Siegel et al. 2005).
Trypanothione participates in a wide range of reactions, including reduction of
dihydroascorbate (Krauth-Siegel & Ludemann 1996), hydroperoxides (pair trypanothione/
tryparedooxin) (Flohé et al. 1999; Flohé et al. 2002) and ribonucleotides (Dormeyer et al. 2001). This
thiol is also involved in the elimination of 2-oxoaldehydes, such as methylglyoxal (Irsch & Krauth-
Siegel 2004; Sousa Silva et al. 2005), a toxic by-product of glycolysis formed in all living cells.
22.. MMeetthhyyllggllyyooxxaall bbiioocchheemmiissttrryy
The first characterization of methylglyoxal’s catabolic enzymes dates back to 1913, with the
identification of a system enabling the conversion of α-oxoaldehydes to α-hidroxyacids, presently
recognised as the glyoxalase system (Dakin & Dudley, 1913a; Dakin & Dudley, 1913b; Neuberg,
1913). Since 1928, methylglyoxal was considered as a key glycolytic intermediate (Neuberg & Kobel,
1928). Twenty years later this perception was abandoned when glutathione was identified as an
indispensable co-factor of the glyoxalase system, keeping in mind that the glycolytic pathway does
not use glutathione (Lohman 1932). Furthermore, D-lactate was found to be this pathway’s product,
instead of the L-lactate glutathione-independent production in muscle cells (Racker 1951). Even
when methylglyoxal was dismissed as a metabolic intermediate, its function and role arouse
scientific community’s interest concerning glyoxalase functions, with methylglyoxal and D-lactate
being detected in a wide range of organisms (Hopkins &
Morgan 1945). The hypothesis that methylglyoxal and the
glyoxalase pathway could be involved in controlling cell
division and cancerogenesis (Szent-Gyorgyi, 1965) enthused
scientific investigation in this field. Presently, we know that
methylglyoxal is present in all cells as a very reactive but
unavoidable product of secondary metabolism.
Methylglyoxal’s reactivity arises from the presence of
two highly reactive carbonyl groups (Figure I.10.). Within biological molecules, amino groups are
the most prevalent nucleophile groups. Being part of proteins, nucleic acids and basic
phospholipids they might be modified by methylglyoxal, justifying its mutagenic and toxic nature.
Figure I.10. Methylglyoxal. (Carbons in grey, hydrogens in white and oxygen atoms in red.)
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Due to its ability to glycate DNA, leading to genomic integrity loss, methylglyoxal is reported as a
mutagenic and genotoxic agent (Migliore et al. 1990, Rahman et al. 1990; Pischetsrieder et al. 1999).
One of the most severe methylglyoxal effects is the glycation of proteins and basic phospholipids,
leading to the formation of advanced glycation end-products (AGE) (Bucala et al. 1993). In proteins,
methylglyoxal’s main targets are arginine and lysine residues, and the derived products are named
MAGE (methylglyoxal-derived advanced glycation end-products) (Gomes et al. 2005a). Moreover,
methylglyoxal is accountable for protein cross-linking. For example, from the reaction of
methylglyoxal with lysine residues a specific AGE can be formed, MOLD (methylglyoxal-lysine
dimers), an AGE present in lens proteins, as well as in diabetic patients (Nagaraj et al. 1996; Frye et
al. 1998).
Methylglyoxal effects have not always been described as hazardous. Indeed, an anti-tumour
activity in vivo was reported (Apple & Greenverg 1967; Conroy 1978; Dianzani 1978), although no
clinical application has been developed for the lack of ability to select the compound’s toxicity.
Recent studies revealed that methylglyoxal enhanced the chaperone function of α-crystallin and
inhibits glycation-mediated pentosidine synthesis that would cause loss of α-crystallin chaperone
function (Puttaiah et al. 2007). Also, it was shown that in yeast, glycation elicits a cellular response
involving heat shock proteins from the refolding chaperone pathway, being the Hsp26p activated
by glycation (Gomes et al. 2008).
22..11.. MMeetthhyyllggllyyooxxaall bbiioossyynntthheessiiss
Methylglyoxal may be synthesised through either enzymatic or non-enzymatic pathways,
depending on the organism (Figure I.11.). In bacteria, it can be produced enzymatically through the
enzyme methylglyoxal synthase (EC 4.2.3.3; Cooper & Anderson 1970). Methylglyoxal can also be
formed from enzymatic reactions involved in the L-threonine metabolism (Ray & Ray 1987; Lyles &
Chalmers 1992); or by the catabolism of ketone bodies, acetoacetate and acetone (Casazza et al. 1984;
Koop & Casazza 1985; Aleksandrovskii 1992). Non-enzymatically, it can be formed through
lipoperoxidation reactions (Esterbauer et al. 1982) or as a by-product of glycolysis from the β-
elimination of phosphate from the triose phosphates dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate (GAP) (Richard 1993). These methylglyoxal biosynthetic systems will
be described in detail.
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Figure I.11. Main routes of methylglyoxal production. This toxic compound is produced both enzymatically (dark-green arrows), from L-threonine and ketone bodies metabolism (Casazza et al. 1984; Lyles & Chalmers 1992); and non enzymatically (light-green arrows), from lipoperoxidation or as a by-product of glycolysis, by β-elimination of the phosphate group from dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP) (Richard 1993). Methylglyoxal synthase is only present in prokaryotes (Hopper & Cooper 1971; Hopper & Cooper 1972). Adapted from Ricardo Gomes, PhD Thesis 2008.
Methylglyoxal synthase (glycerine-phosphate phosphor-lyase, EC 4.2.3.3), first purified from
Escherichia coli and only detected in prokaryotes, catalyses the triose phosphate dihydroxyacetone
phosphate (DHAP) conversion into methylglyoxal. In bacteria, methylglyoxal formation also
constitutes a bypass to glycolysis, as the D-lactate produced through the glyoxalase pathway might
be converted to pyruvate by the enzyme D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase,
EC 1.1.1.28) (Cooper & Anderson 1970). Inorganic phosphate is a common methylglyoxal synthase
allosteric inhibitor. If this compound is present in high concentrations, glycolysis tends occur
through glyceraldehyde 3-phosphate dehydrogenase (GAPDH, D-glyceraldehyde-3-
phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), whose activity is inorganic
phosphate dependent (Hopper & Cooper 1971; Hopper & Cooper 1972). Under conditions of
phosphate starvation, methylglyoxal synthase activity is increased and methylglyoxal arises from
DHAP. This is a regulation mechanism which allows an effective use of inorganic phosphate by
glycolysis, while keeping the production of pyruvate (Cooper 1984). Hence, a glycolysis regulation
role in bacteria was attributed to methylglyoxal synthase, based on intracellular phosphate
availability (Cooper 1984).
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Another methylglyoxal biosynthetic pathway is the L-threonine catabolism, via
aminoacetone catalysed by the enzyme amine oxidase (SSAO, amine:oxygen oxidoreductase,
EC.1.4.3.6) (Lyles & Chalmers 1992). This pathway’s main role is to enzymatically produce glycine
and acetyl-CoA, with aminoacetone as an intermediate. In the presence of low levels of CoA (e.g.
diabetic ketoacidosis, where most CoA is in the form of acetyl-CoA), the production of
aminoacetone from threonine increases, enhancing methylglyoxal production through SSAO
activity (Tressel et al. 1986).
Methylglyoxal is also generated by the oxidation (enzymatic or non-enzymatic) of
acetoacetate by myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC. 1.11.1.7)
(Aleksandrovskii 1992; Kalapos 1999), and by acetone enzymatic oxidation by cytochrome P450
IIE1 (reduced-flavoprotein:oxygen oxidoreductase, EC 1.14.14.1) in a NADPH-dependent two-step
reaction, with acetol as intermediate (Casazza et al. 1984; Koop & Casazza 1985). Ketone bodies
might provide a significant supply of methylglyoxal when under pathological conditions like
ketosis and diabetic ketoacidosis (Turk et al. 2006).
In the main pathway for methylglyoxal biosynthesis, it arises as a by-product of glycolysis.
Methylglyoxal is formed non-enzymatically from the 1,2-enediolate, a common intermediate to
DHAP and GAP, through the irreversible β-elimination of the phosphate group (Richard 1984;
Figure I.11.). At physiological pH, phosphorylated trioses are much more reactive towards the loss
of α-carbonyl protons than the corresponding triose, producing an enediolate phosphate
intermediate, which has a low energy barrier for the phosphate group expulsion. Hence, it is the
substrate deprotonation to an enediolate phosphate intermediate followed by the phosphate group
cleavage that lead to the formation of methylglyoxal (Richard 1993). Triose phosphate isomerase
(TIM, D-glyceraldehyde-3-phosphate aldose-ketose-isomerase, EC. 5.3.1.1) ensures the required
stabilization of the enzyme-bound enediolate phosphate intermediate, reducing substrate
degradation into methylglyoxal, by making the protonation of the enzyme-bound enediolate
phosphate intermediate faster than the phosphate group expulsion (Richard 1991). However, this
non-enzymatic formation of methylglyoxal is unavoidable, with a formation rate estimated to be 0.1
mM per day (Richard 1993).
The overall methylglyoxal formation rate depends on the organism, tissue, cell, metabolism
and physiological conditions. Nevertheless, it seems to be related to the glycolytic flux, confirming
that the glycolytic bypass is the main methylglyoxal biosynthesis pathway (Fareleira et al. 1997;
Martins et al. 2001; Altenberg & Greulich 2004).
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2.1.1 Methylglyoxal biosynthesis in trypanosomatids
In L. infantum, methylglyoxal seems to be formed non-enzymatically, as suggested by its low
concentration in the parasite and the absence of a methylglyoxal synthase gene and activity (Sousa
Silva et al. 2005). This gene seems to be also absent from other trypanosomatids, including T. brucei,
T. cruzi and L. major (Opperdoes & Michels 2008). There are no available studies for the other non-
enzymatic pathways for methylglyoxal biosynthesis in trypanosomatids. Hence, its formation is
commonly considered to occur through the spontaneous phosphate elimination from glycolytic
triose phosphates (Sousa Silva et al. 2005; Opperdoes & Michels 2008).
22..22.. MMeetthhyyllggllyyooxxaall ccaattaabboolliissmm
Methylglyoxal has shown to be a mutagenic, toxic and inhibitor of glycolytic enzymes
(Leocini et al. 1980; Westwood et al. 1997; Lo et al. 1994; Oya et al. 1999). Furthermore, it causes cell
death, when at high concentrations, and cell growth delay at sublethal concentrations (Kalapos
1999; Okado et al. 1996; Ponces Freire et al. 2003; Maeta et al. 2005b). Hence, organisms, unable to
avoid this compound biosynthesis, developed defensive enzymatic mechanisms to catabolise
methylglyoxal.
The glyoxalase system, involving the glyoxalase I (lactoylglutathione lyase; EC 4.4.1.5,
GLO1) and the glyoxalase II (hydroxyacylglutathione hydrolase; EC 3.1.2.6, GLO2) is the main
catabolic pathway for this α-oxoaldehyde. However, other pathways known to lead to
methylglyoxal degradation are: the enzyme aldose reductase (alditol:NAD(P)+ 1-oxidoreductase,
EC.1.1.1.21.) (Vander Jagt et al. 1992); some oxide-reductases and dehydrogenases, taking advantage
of methylglyoxal ability to be oxidised or reduced (Kalapos 1999); α-oxoaldehyde dehydrogenase
(2-oxoaldehyde:NAD(P)+ 2-oxidoreductase, EC. 1.2.1.23) (Monder 1967); aldehyde dehydrogenase
(aldehyde:NAD+ oxidoreductase, EC.1.2.1.3) (Izaguirre et al. 1998); methylglyoxal reductase (D-
lactaldehyde:NAD+ oxidoreductase, EC. 1.1.1.78) (Ray & Ray 1984); and pyruvate dehydrogenase
(pyruvate: dihydrolipoyllysine-residue acetyltransferase-lipoyllysine 2-oxidoreductase, EC. 1.2.4.1)
(Baggetto & Lehninger 1987). Among these enzymes, the glyoxalases and aldose reductase are
considered the main methylglyoxal catabolic systems.
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2.2.1. The glyoxalase pathway
The glyoxalase, named for the belief of being a single enzyme, was discovered in 1913 by
two different simultaneous studies, which identified the conversion of methylglyoxal to lactic acid
in animal tissues (Neuberg 1913; Dakin & Dudley 1913a; Dakin & Dudley 1913b). In 1951, Racker
showed that in fact two enzymes, glyoxalase I and glyoxalase II, are involved in the latter
conversion (Racker 1951).
The glyoxalase pathway has been considered the main catabolic system of 2-oxoaldehydes,
as methylglyoxal (Thornalley 1990), a compound produced in every organism (Figure I.11.). It
comprises the glyoxalase I (S-D-lactoylglutathione methylglyoxal-lyase, EC 4.4.1.5) and the
glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) that convert methylglyoxal to D-
lactate using reduced glutathione as a specific co-factor (Racker 1951; Thornalley 1990). In this
system, methylglyoxal reacts non-enzymatically with glutathione, forming an hemithioacetal
(Vander Jagt et al. 1975; Thornalley 1990; Thornalley 1993). This compound is then isomerised by
glyoxalase I to the thiolester, S-D-lactoylglutathione (SDL-GSH). This thiolester is then hydrolysed
to D-lactate by glyoxalase II (hydroxyacylglutathione hydrolase EC 3.1.2.6), regenerating GSH
(Thornalley 1990; Vander Jagt 1993a).
Although it is clear that the glyoxalase pathway’s main role is the methylglyoxal
detoxification, many studies suggested a connection between the glyoxalase system and other
physiologic processes. As an example, it was reported that immature, proliferating cells and tissues
have a high glyoxalase I activity but a low glyoxalase II activity, whereas in mature differentiated
cells the reverse is observed (Principato et al. 1982), though no causal relationship between the
glyoxalase pathway and cell proliferation was reported. Also, it is known that S. cerevisiae mutants
for glyoxalase I and glyoxalase II are viable, discarding any association between the glyoxalase
system activity and cell survival, except when cells are challenged with methylglyoxal (Bito et al.
1997; Inoue & Kimura 1996). These assumptions support the interest that has been drawn to this
pathway, especially on the association between methylglyoxal, the derived AGE and the
pathogenesis of diabetic complications and neurodegenerative diseases (Thornalley 1993;
Thornalley 1996).
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Figure I.12. Methylglyoxal catabolism by the glyoxalase pathway and aldose reductase. Methylglyoxal is
formed non-enzymatically (n.e.) from glyceraldehyde-phosphate (GAP) or dihydroxyacetone (DHAP)
phosphate and is further catabolised. In the glyoxalase pathway, methylglyoxal reacts non-enzymatically with
reduced glutathione (GSH), forming an hemithioacetal (HTA). This hemithioacetal is then isomerised by
glyoxalase I (GLO1), forming the thiolester S-D-lactoylglutathione, which is subsequently hydrolysed to D-
lactate by glyoxalase II (GLO2), regenerating GSH. Aldose reductase catabolises methylglyoxal to 1,2-
propanediol in a two-steps NADPH-dependent reaction. Adapted from (Vander Jagt & Hunsaker 2003;
Gomes et al. 2005b).
2.2.1.1. Glyoxalase I
Glyoxalase I (GLO1) is present in most organisms, either prokaryotes or eukaryotes, and it
has been characterised at the molecular and kinetic levels in many of them, including mammalian
tissues (Han et al. 1976; Aronsson & Mannervik 1977; Marmstal & Mannervik 1978; Baskaran &
Balasubramanian 1987), plants (Deswal & Sopory 1991; Deswal & Sopory 1999; Norton et al. 1990),
E. coli (Clugston et al. 1998), S. cerevisiae (Marmstal et al. 1979), L. infantum (Vickers et al. 2004; Sousa
Silva et al. 2005) and Plasmodium falciparum (Deponte et al. 2007).
As mentioned, GLO1 catalyses the isomerisation of a hemithioacetal to the thiolester S-D-
lactoylglutathione. This reaction’s mechanism understanding is hampered by the non-enzymatic
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hemithioacetal formation from methylglyoxal and GSH, implying the simultaneous presence of the
three species in equilibrium. Being the dissociation constant for hemithioacetal 3x10-3 M, in the
range of intracellular glutathione concentration, hemithioacetal concentration is noticeably
dependent on GSH concentration (Vander Jagt 1993a). Consequently, under physiological
conditions such as oxidative stress, where GSH concentration is decreased, the glyoxalase pathway
activity may be diminished (Abordo et al. 1999).
GLO1 was kinetically characterised in several organisms. In situ studies of the glyoxalase
pathway in yeast revealed a Km of 0.53 mM and a V of 0.53 x 10-3 mM.s-1 for glyoxalase I towards the
glutathione-derived hemithioacetal (Martins et al. 2001b). Purified GLO1 from S. cerevisiae has an
apparent Km of 0.41 mM (similar to the value obtained from in situ studies) and a kcat/Km of 18.9x103
mM-1.s-1 for the same substrate (Inoue & Kimura 1996). In this organism, GLO1 has two active sites,
most likely catalysing the same isomerization reaction, due to their high structural similarity
(Frickel et al. 2001). Plasmodium falciparum also has a glyoxalase I with two functional active sites,
having similar catalytic activities but different substrate affinities (Deponte et al. 2007). The apparent
Km values for this enzyme, using the hemithioacetal substrate, are 0.016 mM and 0.103 mM, for both
active sites. The recombinant human GLO1 showed a Km of 0.071 mM towards the glutathione-
derived hemithioacetal and a kcat/Km of 0.22x103 mM-1.s-1 (Ridderstrom & Mannervik 1996). Another
isoform of the human enzyme was identified, differing in position 111 with a glutamate instead of
an alanine, showing glyoxalase I activity but different electrophoretic properties (Kim et al. 1995).
Glyoxalase I was also identified in trypanosomatids. GLO1 activity was detected in L. infantum
protein extracts, with a Km of 0.240 mM towards the trypanothione-derived hemithioacetal (Sousa
Silva et al. 2005). This enzyme was also able to isomerise methylglyoxal-glutathione hemithioacetal
with lower affinity (Km of 1.85 mM). The recombinant L. major GLO1 showed similar kinetic
parameters to the human enzyme (Km of 0.032 mM and kcat/Km of 25x103 mM-1.s-1) although towards
the trypanothione-derived hemithioacetal (Vickers et al. 2004). Similarly, the recombinant L.
donovani GLO1 reacts with the hemithioacetal derived from trypanothione with a Km of 0.028 mM
and a specific activity of 5.60x103 nmol.s-1.mg-1 protein (Padmanabhan et al. 2005). This enzyme also
prefers this substrate relatively to the hemithioacetal derived from glutathione (Padmanabhan et al.
2005). In the same study, L. donovani GLO1 was inhibited by the classical human and yeast GLO1
inhibitors: purpurogallin, flavone, quercetin and lapachol.
Structural information of glyoxalase I enzyme is also available through the X-ray structures
from various organisms, including E. coli (EcGLO1; PDB entries 1F9Z, 1FA5, 1FA6, 1FA7, 1FA8 (He
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et al., 2000)), H. sapiens (HsGLO1; PDB entries 1FRO, 1QIN, 1QIP, 1BH5 (Cameron et al., 1997, 1999;
Ridderstrom et al., 1998)) and L. major (LmGLO1; PDB entry 2C21 (Ariza et al., 2006)). Almost all
eukaryotic and prokaryotic glyoxalase I proteins characterised so far are homodimeric molecules
(Cameron et al. 1997; Sukdeo et al. 2004), with each monomer composed of two βαβββ domains and
the residues from each subunit contributing to the active site pocket. However, in yeast (Thornalley
et al. 2003) and plasmodia (Iozef et al. 2003) the enzyme is monomeric.
Glyoxalase I is a metalloprotein, typically with zinc or nickel at the active site. Zn2+-
dependent GLO1 comprise eukaryotic enzymes, as those from H. sapiens and S. cerevisiae (Aronsson
et al. 1978; Ridderstrom et al. 1998). However, nickel-dependent enzymes have been characterised in
several prokaryotes, such as the Ni2+-dependent GLO1 from E. coli, Pseudomonas aeruginosa, Yersinia
pestis and Neisseria meningitides (Sukdeo et al. 2004). Nevertheless, Pseudomonas putida (prokaryotic)
GLO1 is Zn2+-dependent (Saint-Jean et al. 1998) and L. major GLO1 (eukaryotic) was reported to
contain divalent nickel (Ariza et al. 2006). Hence, this classification of glyoxalase I based on the
metal type is not clear.
2.2.1.2. Glyoxalase II
Glyoxalase II (GLO2), a β-lactamase fold-containing enzyme, was purified and characterised
from several mammals (Uotila 1973; Oray & Norton 1980; Ball & Vander Jagt 1981; Principato et al.
1984; Allen et al. 1993), plants (Norton et al. 1990; Maiti et al. 1997), yeast (Talesa et al. 1990b) and L.
infantum (Sousa Silva et al. 2005; Sousa Silva et al. 2008). In some organisms, the metalloprotein
presents a cytosolic and a mitochondrial isoform (Talesa et al. 1988; Talesa et al. 1989; Talesa et al.
1990; Bito et al. 1997; Maiti et al. 1997; Cordell et al. 2004). In mammals, they are both coded by a
single gene and produced by alternative translation initiation of the gene transcripts (Cordell et al.
2004). However, in yeast, cytosolic GLO2 and mitochondrial GLO4 are coded by different genes
(Bito et al. 1997), resulting in proteins with 59.1 % identity. Being mitochondrial GLO1 absent from
rat (Talesa et al. 1988; Talesa et al. 1989) and yeast (Bito et al. 1997), it was suggested that
mitochondrial GLO2 hydrolyses the S-D-lactoylglutathione diffusing or being transported into this
cell compartment. This could constitute a mitochondrial GSH source (Scire et al. 2000), although
GSH import was described (Martensson et al. 1990). In yeast, GLO4 could also catabolise other GSH
thiolesters in the mitochondria (Thornalley 1990; Thornalley 1993; Vander Jagt 1993a).
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Glyoxalase II kinetic analysis was performed in several organisms. In situ measured activity
of yeast GLO2 revealed a Km of 0.32 mM and a V of 0.0172x10-3 mM.s-1 using S-D-lactoylglutathione
as substrate (Martins et al. 2001b). This substrate was hydrolysed by the recombinant purified GLO2
with a Km of 0.112 mM and by the recombinant purified mitochondrial GLO4 with a Km of 0.072
mM (Bito et al. 1999). The kinetic parameters for the recombinant human enzyme were a Km of 0.187
mM and a kcat of 780 s-1 for the glutathione-derived thiolester (Ridderstrom et al. 1996). In
trypanosomatids, the glyoxalase II reacts with thiolesters derived from trypanothione (Irsch &
Krauth-Siegel 2004; Sousa Silva et al. 2005). GLO2 activity was detected in total protein extracts from
L. infantum (Sousa Silva et al. 2005). This enzyme hydrolysed the S-D-lactoyltrypanothione with a Km
of 0.098 mM and could not react with the glutathione-derived thiolester. The recombinant L.
donovani GLO2 revealed a Km of 0.039 mM using S-D-lactoyltrypanothione as a substrate
(Padmanabhan et al. 2006) but had almost no activity in presence of S-D-lactoylglutathione
(Padmanabhan et al. 2006).
Structurally, GLO2 is a monomer containing two domains. The first folds into a four-layered
β-sheet, typical from metallo-β-lactamases, and the second is predominantly α-helical (Cameron et
al. 1999). Structural information of GLO2 is available through the X-ray structures of different
organisms, including human (PDB entry 1QH5 (Cameron et al. 1999)) and Arabidopsis thaliana (PDB
entry 1XM8 (Marasinghe et al. 2005)). GLO2 proteins contain the highly conserved metal binding
motif THXHXDH, common to all known glyoxalases II, including human and Arabidopsis (Cameron
et al. 1999; Crowder et al. 1997; Zhang et al. 2001). The human GLO2 crystal structure revealed the
presence of two Zn2+ ions per molecule in the enzyme’s active site (Cameron et al. 1999). As for A.
thaliana mitochondrial glyoxalase II, this enzyme can incorporate a range of different metal centres,
being the prevalent the Fe(III)Zn(II) centre (Marasinghe et al. 2005). Also, L. infantum GLO2 enzyme
is able to bind either Zn or Fe (Silva et al. 2008), whereas in Trypanosoma brucei glyoxalase II,
sequence analysis seemed to indicate that zinc is the main metal (Irsch & Krauth-Siegel 2004). For
any of the latter GLO2, one of the metal binding sites consists of three histidine residues, a bridging
aspartic acid residue and a bridging water/hydroxide ion, while the second metal binding site
contains two histidine residues, a terminal bound aspartic acid and the same bridging aspartic acid
residue and a water molecule (Cameron et al. 1999).
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2.2.1.3. The glyoxalase pathway in Trypanosomatids
Leishmania was first reported to produce D-lactate as an end-product of methylglyoxal in 1988
(Darling & Blum, 1988). The glyoxalase pathway, considered the main methylglyoxal catabolising system in
trypanosomatids, was shown to be trypanothione-dependent in T. brucei, L. infantum and L. major (Irsch and
Krauth-Siegel 2004; Vickers et al. 2004; Sousa Silva et al. 2005). Both GLO1 and GLO2 are present and active in
L. major, as confirmed by the production of D-lactate in this organism (Opperdoes & Michels 2008). However,
the GLO1 gene is absent from T. brucei, while L. brasiliensis lacks the GLO2 gene (Opperdoes & Michels 2008).
GLO1 and GLO2 were identified in L. donovani. Both recombinant enzymes from this parasite were
biochemically and kinetically studied (Padmanabhan et al. 2005; Padmanabhan et al. 2006), as previously
mentioned. Also L. major GLO1 was identified, kinetically analysed and structurally studied, as formerly
referred (Vickers et al. 2004; Ariza et al. 2006).
In L. infantum, glyoxalase I and glyoxalase II are present and show specificity towards trypanothione
and S-D-lactoyltrypanothione (SDL-T(SH)2), respectively (Sousa Silva et al. 2005) (Figure I.13.). Both enzymes
are active in protein extracts and act sequentially as a pathway, as shown by time-course analysis (Sousa Silva
et al. 2005). Using trypanothione-derived hemithioacetal, the kinetic parameters for both glyoxalases in total
protein extracts were KmGLO1 0.24 mM and KmGLO2 0.073 mM.
The glyoxalase pathway’s potential as a putative therapeutic target in trypanosomatids lies not only
on its importance as a main catabolic route for methylglyoxal in living cells, but also on the differences
relatively to their hosts. The importance of the glyoxalase pathway as a therapeutic target was studied in L.
infantum by modelling and computer simulation (Sousa Silva et al. 2005). The simulation results showed that
the glyoxalase enzymes were poor therapeutic targets, and the inhibition of both GLO1 and GLO2 would
have only a slight to no effect, respectively, on methylglyoxal steady-state concentration. In the same study, it
was also demonstrated that methylglyoxal formation rate and trypanothione concentration would have,
respectively, a direct and inverse proportional effect on methylglyoxal concentration. Despite this conclusion,
this study revealed that LiGLO2 is specific for the trypanothione-derived thiolester, not having activity in the
presence of S-D-lactoylglutathione. LiGLO1 can also react with the hemithioacetal formed from glutathione
and methylglyoxal, although it shows a higher affinity for the trypanothione-derived substrate (Sousa Silva et
al. 2005).
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Figure I.13. Metabolism of methylglyoxal in L. infantum. Methylglyoxal is formed non-enzymatically (n.e.)
from glyceraldehyde-3-phosphate (GAP) or dihydroxyacetone phosphate (DHAP) and is further catabolised
through the glyoxalase pathway. Methylglyoxal reacts non-enzymatically with reduced trypanothione
(T(SH)2), forming an hemithioacetal. This hemithioacetal is then isomerised by glyoxalase I, forming the
thiolester S-D-lactoyltrypanothione, which is subsequently hydrolysed to D-lactate by glyoxalase II,
regenerating T(SH)2 (Barata et al. 2010).
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2.2.2. Aldose Reductase
The aldo-keto reductases (AKRs) is an enzyme superfamily that comprises a wide range of
oxidoreductases, including the aldose reductase (AKR1B; alditol:NAD(P)+ oxidoreductase, EC
1.1.1.21) and aldehyde reductase (AKR1A; aldehyde:NAD(P)+ oxidoreductase, EC 1.1.1.2) (Jez &
Penning 2001). Members of AKR were found in both prokaryotes and eukaryotes, from bacteria and
archaebacteria to protozoa and fungi, from invertebrates to invertebrates and plants, suggesting
that it is a very ancient superfamily (Jez et al. 1997).
Aldose reductase, the first enzyme of the polyol pathway (Burg et al. 1996), catalyses the
NADPH-reduction of a wide range of aldehydes to polyols (Ginsburg & Hers 1960; Hers 1956),
assuming a detoxification role. The most known reaction catalysed by aldose reductase is the
reduction of D-glucose to sorbitol in a NADPH-dependent reaction, which is then converted to D-
fructose by the NAD+-dependent sorbitol dehydrogenase (EC 1.1.1.14; Jeffery & Jornvall 1983;
Leissing & McGuiness 1983). Hence, aldose reductase is usually linked to diabetes, as the polyol
pathway activity increases upon hyperglycaemia (Gonzalez et al. 1984a; Gonzalez et al. 1984b),
leading to cellular damage and NADPH/NAD+ depletion (Yabe-Nishimura 1998). Therefore,
several aldose reductase inhibitors, envisioning therapeutic possibilities, are available (Ramasamy et
al. 1997; Yabe-Nishimura 1998; Iwata et al. 2006).
Although D-glucose catabolism has impelled most of the aldose reductase studies, this is a
poor substrate for this enzyme, with a Km of 70 mM and a kcat/Km of 0.015 mM-1.s-1, when compared
to other physiological substrates (Vander Jagt et al. 1990; Vander Jagt et al. 1992). Aldose reductase
has a much higher affinity towards methylglyoxal (Km of 0.008 mM and kcat/Km of 3.0 mM-1.s-1)
(Vander Jagt et al. 1992), which is reduced to acetol, in a NADPH-dependent reaction (Figure I.12.).
Being also an aldose reductase substrate, acetol is reduced in a NADPH-dependent reaction to D-
1,2-propanediol (Vander Jagt et al. 1992).
Methylglyoxal promotes a dose- and time-dependent increase in aldose reductase mRNA,
protein level and enzymatic activity (Yabe-Nishimura et al. 2003). In S. cerevisiae, aldose reductase is
coded by the GRE3 gene (stress response gene, YHR104W). Its expression is up-regulated osmotic
and oxidative stress to efficiently eliminate the extra methylglyoxal produced under these
conditions (Aguilera & Prieto 2001).
Most aldose reductase available structures are from the human enzyme, most probably due
to its importance in diabetes. The first human aldose reductase structure was solved in 1992 (PDB
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entry 1ADS (Bhorani et al. 1992)). Since then, several others became available, including structures
with a broad range of inhibitors and small molecules. Besides the human enzyme, there are also
accessible other aldose reductase structures from other organisms, including the protozoan Giardia
lamblia (PDB entry 3KRB (Seattle Structural Genomics Center for Infectious Disease, to be
published)) and the mammal Sus scrofa (PDB entry 1AH4 (Urzhumtsev et al. 1997)). As other
members of the AKR superfamily, structurally known aldose reductases are monomeric (α/β)8-
barrel proteins which bind NADPH to reduce an array of substrates at an active site containing a
tyrosine, a lysine, an aspartate and a histidine (Ye et al. 2001).
2.2.2.1. Aldose reductase in Trypanosomatids
The first known study on aldose reductase in trypanosomatids reports the identification of a
developmentally regulated acidic polypeptide of Mr= 32000 in L. major, with great sequence
similarity (40-46 %) to the reductase superfamily (Samaras et al. 1988). In 1989, a NADPH-
dependent methylglyoxal reductase was reported to be active in L. donovani (Goshal et al. 1989). The
authors considered this enzyme specific for methylglyoxal being different from the less specific
aldehyde reductases. In 1995, another protein identified as aldehyde reductase from L. donovani was
referred to catabolise acetaldehyde to ethanol in the presence of NADPH (Keegan & Blum 1995).
Recently, it was reported the purification and kinetic analysis of a member of the AKR family in L.
donovani (Rath et al. 2009). Although the authors classify this enzyme as an aldose reductase, it
shares 95 % homology with L. major prostanglandin-f2-alpha-synthase and the structural model
presented was based on the T. brucei brucei prostaglandin-f-synthase. As mentioned, the AKR
superfamily includes a wide range of proteins, being the prostaglandin-f2-alpha-synthase a close
smaller relative. Structural information of Leishmania aldose reductase is still unavailable.
Aldose reductase activity was found in L. infantum promastigotes total protein extracts
(Sousa Silva, data not published). This enzyme reduced methylglyoxal using NADPH with a KmMG
of 0.78 mM and a KmNADPH of 0.0033 mM (Sousa Silva, data not published), similar to other known
aldose reductases (Vander Jagt et al. 1992; Vander Jagt et al. 1993b).
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33.. AAiimmss aanndd ssccooppee ooff tthhiiss wwoorrkk
Enzymes tend to develop differences along evolution and across-organisms. Leishmania infantum, like
other trypanosomatids, has relevant biochemical characteristics which distinguishes it from other eukaryotes,
being one of the most outstanding the functional replacement of glutathione by trypanothione. Consequently,
all glutathione-dependent enzymes are replaced by trypanothione-dependent ones. Understanding these
enzymes is obviously crucial for a better insight into this parasite’s very unique metabolism.
This Ph.D. project’s scope was the characterization at the kinetic and molecular levels of the enzymes
involved in methylglyoxal catabolism in L. infantum, namely the glyoxalase pathway enzymes and the aldose
reductase.
The first aim was the kinetic and X-ray structural analysis of the recombinant enzymes of the L.
infantum glyoxalase pathway, glyoxalase I and glyoxalase II, and its comparison to other studied homologues,
targeting the understanding of each enzyme’s trypanothione specificity. Hence, the particular steps were to
identify and isolate genes, produce both recombinant proteins, solve their tridimensional structures by X-ray
crystallography and, in parallel, determine their kinetic parameters in the presence of different substrates. The
determination of L. infantum glyoxalase I and glyoxalase II structures, combined with enzyme kinetics, is
important for insight into the substrate binding, understanding the catalytic mechanism and the importance
of the pathway. The study of each of the latter enzymes had two different purposes: the detailed metal
analysis of L. infantum glyoxalase I and the substrate-binding analysis through mutation of L. infantum
glyoxalase II.
The second goal of this work was the de novo kinetic and structural study of L. infantum aldose
reductase, the second most important catabolic pathway for methylglyoxal. Following identification of the
gene coding for this enzyme, the aim was to purify the correspondent protein in significantly high yields to
pursue the enzyme kinetics and structure determination, an advantage for the understanding of this parasite’s
detoxification mechanism for methylglyoxal.
Barata, L., Sousa Silva, M., Schuldt, L., Costa, G., Tomás, A. M., Ferreira, A. E. N., Weiss, M. S., Ponces
Freire, A., Cordeiro, C. (2010) Cloning, expression, purification, crystallization and preliminary X-ray
Diffraction Analysis of Glyoxalase I from Leishmania infantum. Acta Crystallographica Section F, 66, 571–574.
Manuscript on LiGLO1 structure and metal analysis in preparation.
CChhaapptteerr IIII GGllyyooxxaallaassee II ffrroomm
LLeeiisshhmmaanniiaa iinnffaannttuumm
CChhaapptteerr IIII
3311
11.. SSuummmmaarryy
Glyoxalase I (GLO1) is the first of the two enzymes of the glyoxalase pathway,
catalysing the formation of S-D-lactoyltrypanothione from the hemithioacetal non-
enzymatically formed from methylglyoxal and reduced glutathione. In trypanosomatids,
parasitic protozoa that cause mammal diseases such as leishmaniasis and trypanosomatiasis,
glutathione is replaced by trypanothione as a cofactor for glyoxalase I. GLO1 from L.
infantum (LiGLO1) was cloned, over-expressed in E. coli, purified and crystallised. Two
crystal forms were obtained: a cube-shaped and a rod-shaped one. While the cube-shaped
form does not diffract X-rays at all, the rod-shaped form exhibits diffraction to about 2.0 Å
resolution. The crystals belong to space group P21212 with the unit cell parameters a= 130.03
Å, b= 148.51 Å, c= 50.63 Å and three dimers of the enzyme per asymmetric unit.
A metal cofactor is essential for GLO1 activity. Typically, eukaryotic GLO1 contains
Zn2+ at the active site and prokaryotic GLO1 contain Ni2+. However, a general rule to define
the metal-type according to the taxonomic group cannot be formulated since there are some
exceptions. By Inductively-Coupled Plasma (ICP) emission, X-ray fluorescence and X-ray
anomalous diffraction it could be undoubtedly established that the major metal occupying
the LiGLO1 active site is Zn2+. This finding is in stark contrast to the reported presence of
Ni2+ in the closely related LmGLO1 enzyme.
LiGLO1 was active and able to use both glutathione and trypanothione
hemithioacetals as substrate, although the latter was preferred. Hence, LiGLO1 differs from
the human enzyme counterpart in active site structure and substrate specificity. The 2.0 Å
resolution LiGLO1 structure reported in this work, together with the solved L. infantum
glyoxalase II (LiGLO2), will provide the basis for a better understanding of the glyoxalase
pathway in trypanosomatids and of the evolutionary pressure towards trypanothione
specificity.
CChhaapptteerr IIII
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22.. IInnttrroodduuccttiioonn
The enzyme glyoxalase I (GLO1; lactoylglutathione lyase; EC 4.4.1.5) is the first of
two enzymes involved in the thiol-dependent glyoxalase pathway (Thornalley 1990).
Together with the glyoxalase II (GLO2; hydroxyacylglutathione hydrolase, EC 3.2.1.6), it is
essential for the detoxification of methylglyoxal, a toxic and mutagenic (Marinari et al. 1984;
Lo et al. 1994) non-enzymatically formed by-product of glycolysis (Thornalley et al. 1996).
The hemithioacetal spontaneously formed by methylglyoxal and glutathione is sequentially
isomerised by glyoxalase I to S-D-lactoylglutathione and then hydrolysed by glyoxalase II to
D-lactate and glutathione (see figure I.13. from this thesis). Trypanosomatids, the causative
agents of mammal diseases, are exclusive concerning the use of trypanothione (N1,N8-
bis(glutathionyl)spermidine, T(SH)2) instead of glutathione (γ-L-glutamyl-L-
cysteinylglycine; GSH) in many metabolic processes. The glyoxalase pathway is no
exception, although the glyoxalase I from trypanosomatids can also use the glutathione-
methylglyoxal hemithioacetal (Vickers et al. 2004; Sousa Silva et al. 2005).
Eukaryotic (Cameron et al. 1997) and prokaryotic (Sukdeo et al. 2004) GLO1 are
typically homodimeric proteins with subunits of about 140 residues and a molecular weight
of about 18 kDa. Exceptions include the yeast (Thornalley 2003) and plasmodia (Iozef et al.
2003) GLO1 enzymes, which are monomeric, albeit coded by a duplicated gene in both
cases. Glyoxalase I three dimensional structural information is available for E. coli (EcGLO1;
PDB entry 1F9Z (He et al. 2000)), human (HsGLO1; PDB entry 1FRO (Cameron et al. 1997))
and L. major (LmGLO1; PDB entry 2C21 (Ariza et al. 2006)). All of these structures constitute
homodimeric enzymes, where each subunit is composed of two βαβββ domains and the
active site is located at the interface between the two subunits.
GLO1 belongs to two superfamilies: the βαβββ superfamily, characterised by
proteins with a cleft, which in GLO1 harbours the metal-binding and active site; and the
VOC (vicinal oxygen chelate) superfamily, which comprises enzymes featuring metal centre
co-ordination by substrate oxygen atoms (Sukdeo et al. 2004). The metal centre is essential
for the enzymes of both families (Sukdeo et al. 2004).
According to the metal-content, GLO1 have been divided in two groups: the Zn2+-
dependent GLO1, comprising eukaryotic GLO1, as those from H. sapiens and S. cerevisiae
CChhaapptteerr IIII
3333
(Aronsson et al. 1978; Ridderstrom et al. 1996), and Ni2+-dependent ones, including
prokaryotic enzymes from E. coli, Pseudomonas aeruginosa, Yersinia pestis and Neisseria
meningitides (Sukdeo et al. 2004). However, this division by metal-type is not accurate, since
the prokaryotic Pseudomonas putida GLO1 is Zn2+-dependent (Saint-Jean et al. 1998) while the
eukaryotic L. major GLO1 (LmGLO1) contains divalent nickel at the active site (He et al.
2000).
Here, we report the cloning, expression, purification, crystallization and preliminary
X-ray diffraction analysis of LiGLO1. In addition, the refined structure of LiGLO1 is
described, as well as its detailed kinetic and metal analysis. X-ray anomalous scattering
methods, fluorescence and Induced Coupled Plasma (ICP) analysis were used to elucidate
the nature of the metal in the LiGLO1 metal binding active site. LiGLO1 and LmGLO1
appear to have a different metal specificity although they share 97 % amino acid sequence
identity. In LmGLO1, 0.9 mol of nickel and 0.1 mol of zinc per mol of protein were detected
by atomic absorption spectrophotometry (Vickers et al. 2004), indicating that the
homodimeric protein active sites might not tightly bind the metal cofactor (Vickers et al.
2004; Clugston et al. 1998). Although the predicted metal-binding residues are conserved in
these two enzymes, zinc is the main metal present in the in LiGLO1’s active site. L. major and
L. infantum cause different forms of leishmaniasis, cutaneous and visceral respectively, being
very interesting to find significant differences in their respective biochemistry, most
probably related to protein evolution.
The studies presented in this work, together with the LiGLO2 solved structure and
kinetic analysis (Silva et al. 2008), will allow a complete assessment of the L. infantum
glyoxalase pathway and provide insights into the evolutionary path leading to glyoxalase I
metal and substrate specificity.
CChhaapptteerr IIII
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33.. MMaatteerriiaallss aanndd MMeetthhooddss
33..11.. CClloonniinngg aanndd eexxpprreessssiioonn ooff LLiiGGLLOO11
The LiGLO1 gene (GenBank accession no. DQ294973) was amplified from L. infantum
genomic DNA. The PCR product was cloned (forward primer 5’-
CCGCGCACATATGCCGTCTCGTCGTAT-3’, containing the NdeI restriction site
(underlined), and reverse primer 5’-CACCGCTCGAGTTACGCAGTGCCCTGCTC-3’,
containing the XhoI restriction site (underlined) immediately after the stop codon) into the
NdeI/XhoI-digested expression vector pET28a (Novagen), which was then transformed into
Escherichia coli BL21-CodonPlus (Stratagene). The correct cloning of the open reading frame
was confirmed by sequencing. The final protein sequence was preceded by
MGSSHHHHHHSSGLVPRGSH, containing the His6-tag (bold) and the thrombin cleavage
site (underlined). For over-expression of the N-terminally His6-tagged LiGLO1, E. coli BL21-
Codon Plus transformants were grown in LB medium containing 50 µg ml-1 kanamycin and
34 µg ml-1 chloramphenicol at 37°C. Expression of His6-tagged LiGLO1 was induced at the
culture OD600 of 0.6 with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 3 h at 37°C.
Cells were harvested by centrifugation at 7800 g for 30 min at 4°C. Cell pellets were frozen at
-20°C.
33..22.. PPuurriiffiiccaattiioonn ooff LLiiGGLLOO11
The cell pellet was suspended in 10 ml of buffer A (50 mM Bis-Tris pH 6.3, 200 mM
NaCl, all chemicals were obtained from Carl Roth, Karlsruhe, Germany) per g of cells and
lysed by sonication for 10 minutes at 35 % power and 0.4s pulses with 0.9s breaks between
the pulses at 4°C. The soluble fraction obtained by centrifugation at 38000 g for 40 min at 4°C
was filtered through a 0.22 µm membrane, before being applied to affinity chromatography
NTA resin charged with cobalt (Co-NTA) or nickel (Ni-NTA), resulting in two batches of
protein, identified as LiGLO1 purified using Co-NTA and Ni-NTA, respectively, for
purification at room temperature. The fusion protein, with an N-terminal tail of six histidine
residues and a thrombin cleavage site was eluted in buffer A supplemented with 500 mM
imidazole. The N-terminal His6-tag was cleaved using the Thrombin Cleavage Kit
CChhaapptteerr IIII
3355
(Novagene) and optimised cleavage conditions (1:200 thrombin dilution, overnight, at 20°C).
The cleaved protein was directly applied to a gel filtration column (Superdex S75 16/60, GE
Healthcare) equilibrated with buffer A for further purification. The purity of the
recombinant enzyme was estimated by SDS-PAGE. To confirm the identity of the expressed
protein, peptide mass fingerprinting by MALDI-FTICR-MS (Shevchenko et al. 2006) was
performed by Gonçalo da Costa at the FTICR and Advanced Proteomics Laboratory, FCUL,
Portugal. LiGLO1 was concentrated to 15 mg ml-1 in buffer A using a Vivaspin15 (molecular
weight cutoff 10000) concentrator (Sartorius Stedim Biotech). Protein concentration was
determined using a Peqlab Biotechnologie GmbH NanoDrop® ND-1000 Spectrometer at 280
nm, assuming an estimated LiGLO1 extinction coefficient of 20400 M-1 cm-1, as calculated by
ProtParam tool (Gasteiger et al., 2005) for a protein molecular weight of 18.2 kDa. Buffer A
was optimised by ligand-assisted monitoring of thermal protein unfolding in a ThermoFluor
experiment (Ericsson et al. 2006). 5 µl of 50 × Sypro Orange (fluorophore, Invitrogen), 5 µl of
1 mg/ml LiAKR and 40 µl of each buffer were added to the wells of a 96-well thin-wall PCR
plate (Bio-Rad). Fluorescence was detected while heating the plate from 4 to 95 °C in
increments of 0.5 °C using a ThermoFluor ICycler IQ thermal cycler (Bio-Rad). Two screens
were performed: a pH versus NaCl concentration screen and an additives screen, including
various compounds (nucleotides, metal ions, salts and others) supplemented to the basis
buffer 50 mM Tris-HCl pH 8.5.
33..33.. MMeettaall aannaallyyssiiss bbyy IICCPP
LiGLO1 metal analysis was performed by atomic emission spectrometry Inductively-Coupled
Plasma (ICP) (Faires et al. 1993). LiGLO1 (50 µg/ml) was analysed for nickel, cobalt and zinc using a
Jobin Yvon-Ultima ICP spectometer available at the Atomic Emission Spectrometry Service (ICP),
Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal.
33..44.. CCrryyssttaalllliizzaattiioonn
Initial crystallization screening was performed at the High Throughput
Crystallization facility at the EMBL-Hamburg Outstation (Mueller-Dieckmann, 2006) using
commercially available crystal screens and the sitting-drop vapour-diffusion method. The
drops consisted of 0.5 µl of protein solution at a protein concentration of 15 mg/ml and 0.5
µl of reservoir solution, and were equilibrated over 80 µl reservoir solutions using 96-well
CChhaapptteerr IIII
3366
format Greiner plates. Two crystal forms were obtained from two different crystallization
conditions: conditions B1 (70 % (v/v) MPD, 100 mM MES-Na salt pH 6.5) and B2 (70 %
(v/v) MPD, 100 mM Tris-HCl pH 8.5) of JBScreen Classic 8 from Jena Bioscience (Jena,
Germany). Crystals of form 2 were then further improved by applying the hanging-drop
vapour-diffusion method using the tools from Nextal Biotechnology. Drops were prepared
by mixing 2 µl protein solution with 2 µl reservoir solution containing 62 % (v/v) MPD (2-
methyl-2,4-pentanediol) and 100 mM Tris-HCl pH 8.5, and equilibrated over 1 ml reservoir
solution.
33..55.. DDaattaa ccoolllleeccttiioonn aanndd pprroocceessssiinngg
For diffraction experiments, the crystals mounted into nylon loops were directly
flash-cooled in the cryostream without any additional cryoprotectant. The cube-shaped
crystals (Figure II.1a.) did not show any X-ray diffraction at beamline ID23-2 at the
European Synchrotron Research Facility (ESRF, Grenoble, France). The initial rod-shaped
crystals from the second condition, however, (Figure II.1b.) diffracted to 3.0 Å at the same
beamline and a complete diffraction data set was collected. A 2.1 Å resolution complete
native data set was collected from an optimised form 2 single crystal at EMBL-beamline X12
(Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany), equipped with a MAR
Mosaic CCD 225 detector. Data were processed and scaled using XDS (Kabsch et al. 1993).
LiGLO1 crystals from the batches purified using Co-NTA and Ni-NTA, respectively,
were used for the analysis of the metal composition of the active site. The four energies for
data collection (9950eV, 8600eV, 8000eV and 7500eV) were determined from the theoretical
absorption curves for zinc, nickel and cobalt (Figure II.5.), respectively. For each purification
batch (Co-NTA and Ni-NTA) two crystals from the same crystallization drop were used for
the analysis. For crystal 1 four datasets were collected with stepwise decreasing energy
(9950eV, 8600eV, 8000eV and 7500eV). For crystal 2 data collection was performed with
stepwise increasing energies (7500eV, 8000eV, 8600eV and 9950eV). The respective crystal-
to-detector distances were adjusted for each energy, to obtain an equivalent resolution range
at the edge of the detector.
Indexing, integration and scaling of the data was performed using XDS and XSCALE
(Kabsch et al. 1993). The crystals belong to the orthorhombic space group P21212 and all
CChhaapptteerr IIII
3377
diffracted to at least a resolution of 2.25 Å. The R factors Rr.i.m. (redundancy-independent
merging R factor) and Rp.i.m. (precision-indicating merging R factor) (Weiss 2001) were
calculated using RMERGE (available from: www.embl-
hamburg.de/~msweiss/projects/rmerge.f or from MSW upon request).
The details of data collection and processing are summarised in Table II.1. and Table
II.2. Intensities were converted to structure factor amplitudes using the program
TRUNCATE (French & Wilson 1978; Collaborative Computational Project, Number 4, 1994).
The optical resolution of the data sets was calculated with SFCHECK (Vanguine et al. 1999).
33..66.. CCrryyssttaall ssttrruuccttuurree ssoolluuttiioonn
The three-dimensional crystal structure of LiGLOI was solved by molecular
replacement using the program MOLREP (Vagin & Teplyakov 1997) and one polypeptide
chain of glyoxalase I from L. major (PDB entry 2C21 (Ariza et al. 2006)) as a search model.
Three protein dimers per asymmetric unit were refined against a native data set of 2.1 Å in
space group P21212 (Table II.1. and II.2.). The model was adjusted using COOT (Emsley &
Cowtan 2004) and refinement was performed with Refmac5 (Murshudov et al. 1997). The
final refinement statistics for the native data are given in Table II.1. All molecular graphics
images were generated using the UCSF Chimera (Pettersen et al. 2004). For the datasets of
the metal analysis, the structures were determined by difference Fourier maps phased using
the atomic coordinates of the polypeptide chains from the native structure. One round of
refinement using Refmac5 (Murshudov et al. 1998) was performed, including 20 cycles of
rigid body refinement and 40 cycles of restrained refinement.
33..77.. AAnnoommaalloouuss ddiiffffeerreennccee mmaappss
The program FFT (Bailey 1994) was used to calculate an anomalous Fourier map.
MAPMASK and PEAKMAX (Bailey 1994) were used to search for peaks in the electron
density map above a threshold of 0.1. The atomic coordinates of native LiGLO1, which are
close to the anomalous peaks from the PEAKMAX output, were identified with WATPEAK
(Bailey 1994). The asymmetric unit of GLO1 contains three dimers, which are named AB, CD
CChhaapptteerr IIII
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and EF, respectively. Each dimer exhibits two metal binding sites, termed AB1 and AB2,
respectively (accordingly for the remaining two dimers).
33..88.. OOccccuuppaanncciieess ooff mmeettaall bbiinnddiinngg ssiitteess
The values of the anomalous components ∆f” for the elements zinc, nickel, cobalt and
sulfur for the four energies of data collection were taken from the internet site of the
Biomolecular Structure Center at the University of Washington, Seattle, USA
(http://skuld.bmsc.washington.edu/scatter/AS_periodic.html). To specify the values for
the different metal ions at different energies, the nomenclature used here includes the name
of the element E and the respective value of the energy used for data collection, resulting in
∆f”(E,eV) (e.g. ∆f”(Zn,9950)).
Scaling of the occupancy of the metal binding sites is performed intrinsically against
the anomalous signal of sulfur. Within each dimer the anomalous difference peak with the
highest sigma value σmax at a sulfur position is considered to have occupancy of 100 %.
Considering the ∆f” value of sulfur at the corresponding energies ∆f”(S,eV) (Table II.4.), a
scaling factor SXY,eV for each dimer and each energy is generated by
SXY,eV= σmax,eV/∆f”(S,eV).
Each metal binding site (generally XY; more precisely AB1, AB2, CD1, CD2, EF1 and
EF2) is assumed to have zinc, nickel and cobalt ions bound at an unknown ratio. All these
metal ions contribute to the anomalous signal and the contribution depends on the
occupancy (described by the variables cZn, cNi and cCo, respectively) and on the
corresponding ∆f” values for the different metal ions at different energies. Hence, the sigma
value σXY for the observed anomalous difference peak can be explained by the linear
combination
σXY,eV = cZn * ∆f”(Zn,eV) * SXY,eV + cNi * ∆f”(Ni,eV) * SXY,eV + cCo * ∆f”(Co,eV) * SXY,eV
For each binding site XY, four analogue equations at different energies result and the
values cZn, cNi and cCo can be solved. This calculation was developed by Linda Schuldt and
was implemented in the program LINSOLVE, specifically developed for this work by Gerrit
Langer, at the EMBL-Hamburg, Germany.
CChhaapptteerr IIII
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33..99.. LLiiGGLLOO11 KKiinneettiicc AAnnaallyyssiiss
LiGLO1 was kinetically analysed. For all assays, oxidised trypanothione (Bachem)
was reduced using dithiothreitol (Sigma) immediately before the reaction, as described
(Sousa Silva et al. 2005). High-purity methylglyoxal was used in these assays. Methylglyoxal
was prepared by acid hydrolysis of methylglyoxal 1,1-dimethyl acetal (Sigma-Aldrich),
followed by fractional distillation under reduced pressure in a nitrogen atmosphere, as
described previously (Sousa Silva et al. 2005).
Methylglyoxal was added in excess (3.34 mM in a reactional system of 2 mL), and the
hemithioacetal concentration was calculated considering the dissociation constant 3.0 mM (Vander
Jagt et al. 1972). The activity assay of LiGLO1 (purified using Co-NTA) was undertaken as previously
described (Sousa Silva et al. 2005). Hemithioacetal concentrations between 0.05 and 0.5 mM were
prepared from trypanothione (T(SH)2) and methylglyoxal, whereas for the glutathione (GSH)-derived
hemithioacetal concentrations between 0.1 and 2 mM were used. Thiolesters hydrolysis were
followed at 240 nm. All reactions were performed at 30 °C in 2 mL of 100 mM potassium phosphate
buffer pH 7.4, on an Agilent HP 8453 diode array spectrophotometer (with temperature control and
cuvette stirring). Km values for both substrates were determined using the HYPER32 software (J.S.
Easterby, Univ. of Liverpool, UK) and the limiting rate was calculated by time-course analysis using
an in-house developed software (Ferreira, A. E. N.).
44.. RReessuullttss aanndd ddiissccuussssiioonn
44..11.. TThhee LL.. iinnffaannttuumm ggllyyooxxaallaassee II ggeennee aanndd ddeedduucceedd pprrootteeiinn
A BLAST search carried out on GeneDB (Hertz-Fowler et al. 2004) for the L. infantum
genome revealed only one putative lactoylglutathione lyase-like gene, with 426 bp in length,
at chromosome 35. The sequence was confirmed by sequencing in both directions, after
complete LiGLO1 gene was amplified from genomic DNA of clone
MHOM/MA67ITMAP263. The LiGLO1 sequence (GenBank acc. nr. DQ294973) shows 96 %
identity to L. donovani (GenBank acc. nr. AY739896) and 95 % identity to L. major (GenBank
acc. nr. AY604654) and 33 % identity to human glyoxalase I (GenBank acc. nr. D13315). The
deduced protein was identified as a lactoylglutathione lyase, with 141 amino acid residues.
The predicted molecular mass of 15.7 kDa is similar to L. major and L. donovani enzymes, but
smaller than the 43.0 kDa human glyoxalase I. The isoelectric point (pI) of L. infantum
CChhaapptteerr IIII
4400
glyoxalase I, calculated from the protein sequence, is 4.7. This value is similar to the pI value
of 5.0 for L. donovani (Padmanabhan et al. 2005), 4.9 for L. major (Vickers et al. 2004) and 4.8-
5.0 reported for human glyoxalase I (Ridderstrom et al. 1996).
44..22.. RReeccoommbbiinnaanntt LLiiGGLLOO11 oovveerr--eexxpprreessssiioonn aanndd ppuurriiffiiccaattiioonn
The pET-28a expression vector carrying the LiGLO1 gene was transformed into E. coli
BL21 cells, and over-expression conditions for His6-LiGLO1 were optimised using small-
scale growth cultures. Purification buffer was optimised by ThermoFluor, an assay based on
monitoring the thermal induced unfolding of a protein in presence of several buffers by
measuring the temperature midpoint of the protein unfolding transition (Tm) (Pantoliano et
al. 2001). The results showed that the protein was most stable in 50 mM Bis-Tris pH 6.3 with
200 mM NaCl (data not shown). LiGLO1 purification was performed using a cobalt- and
nickel-affinity chromatography, to address metal identity in the enzyme, with a further gel
filtration step. The protein was about 95 % pure as estimated by SDS–PAGE. The total
purification process yield was about 20 mg of protein per litter of culture, for both
purifications using Co-NTA and Ni-NTA. LiGLO1 is a dimeric protein in solution as
confirmed by preliminary Static Light Scattering (SLS), based on measuring the average
intensity of light scattered by a protein solution of defined concentration (Kratochvil 1987).
44..33.. CCrryyssttaallss,, ddaattaa ccoolllleeccttiioonn aanndd pprroocceessssiinngg
The crystallization behaviour was observed to be similar to the enzyme from L. major
(Ariza et al. 2005), as it resulted in two crystal forms (Figure II.1.). The optimised rod-shape
crystals (Figure II.1c.) grew within 3 days at 20°C to maximum dimension 0.50x 0.15x0.15
mm3. The initial rod-shaped form 2 crystals (Figure II.1b.) diffracted to about 3.0 Å
resolution, while the optimised ones (Figure II.1c.) diffracted X-rays to beyond 2.1 Å
resolution using synchrotron radiation.
CChhaapptteerr IIII
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Figure II.1. Crystals of LiGLO1. (a) Form 1 crystals with dimensions of approximately 0.1x0.1x0.1
mm3. Did not exhibit any diffraction. (b) Initial form 2 crystals with dimensions of approximately
0.15x0.03x0.03 mm3. Diffracted to about 3.0 Å resolution. (c) Improved form 2 crystals with
dimensions of approximately 0.5x0.15x0.15 mm3. Diffracted to about 2.0 Å resolution.
Two complete diffraction data sets were collected (Table II.1.). The crystals belong to
space group P21212, with unit-cell parameters a=130.03 Å, b=148.51 Å and c=50.63 Å. The
Matthews coefficient VM (Matthews 1968) and solvent content, calculated based on a subunit
molecular weight of 18.2 kDa (predicted from the sequence), estimates the most likely
asymmetric unit content to be either four or six molecules. Assuming the presence of three
dimers per asymmetric unit, the Matthews coefficient is 2.2 Å3 Da-1and the solvent content
45.3 % (Kantardjeff & Rupp 2003; Matthews 1968). For two dimers the respective values
would be 3.4 Å3 Da-1and 63.6 %. A detailed examination of the self-rotation function
revealed non-crystallographic peaks on the κ = 180° section (Figure II.2.). It appears to be the
case that at least two additional twofold axes are present on this section and that all of them
lie in the xy-plane. However, without knowledge of the structure, the self rotation function
does not really allow determination of the number of dimers per asymmetric unit with
certainty.
CChhaapptteerr IIII
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Table II.1. Data collection, processing and refinement statistics. The values in parenthesis are for
highest resolution shell. Data were processed using XDS, RMERGE, TRUNCATE and SFCHECK
(Kabsch 1993; Bailey 1994; Vagin & Teplyakov 1997; Weiss 2001). Structure refinement was performed
with Refmac5 (Bailey 1994; Murshudov et al. 1997). Ramachandran plot was produced using
PROCHECK (Kleywegt & Jones 1996; Laskowski et al. 1993).
Data collection and processing: Beamline X12 (EMBL, DESY) Detector MARCCD 225mm Wavelength (Å) 1.0000 Crystal-detector distance (mm) 200 Rotation range per image (°) 0.5 Total rotation range (°) 200 Resolution range (Å) 50.0–2.10 (2.20-2.10) Space group P21212 Unit cell parameters, a, b, c (Å) 130.03, 148.51, 50.63 Mosaicity (°) 0.116 No. of reflections 474893 No. of unique reflections 110729 Redundancy 4.3 (4.2) I/σ(I) 12.8 (2.2) Completeness (%) 99.8 (98.9) Rmerge (%) 1) 9.7 (77.8) Rr.i.m. (%) 2) 10.4 (83.1) Rp.i.m. (%) 3) 3.6 (28.9) Overall B-factor from Wilson plot (Å2) 32.3 Optical resolution (Å) 1.65 Refinement statistics: R factor (%) 18.1 Rfree fator (%) 21.9 No. of atoms Protein 6860 MPD/ Na+/ Zn2+/ water 5/ 2/ 6/ 285 R.m.s. deviations bond lengths (Å) bond angles (°)
0.015 1.701
Ramachandran plot most favoured (%) 95.1 additionally allowed (%) 4.7
1) Rmerge= ΣhklΣi | Ii(hkl) - <I(hkl)> | / ΣhklΣi Ii(hkl), where Ii(hkl) is the intensity of the observation i of the reflection hkl. 2) Rr.i.m= Σhkl (N/(N-1))1/2
Σi | Ii(hkl) - <I(hkl)> | / ΣhklΣi Ii(hkl), where Ii(hkl) is the intensity of the observation i of the reflection hkl and N is the redundancy of the reflection hkl. 3) Rp.i.m= Σhkl (1/(N-1))1/2
Σi | Ii(hkl) - <I(hkl)> | / ΣhklΣi Ii(hkl), where Ii(hkl) is the intensity of the observation i of the reflection hkl and N is the redundancy of the reflection hkl.
CChhaapptteerr IIII
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Figure II.2. Self-rotation function of the LiGLO1 native data. Shown is the κ = 180° section indicating
the relative locations of the twofold symmetry axes. The peaks originating from the crystallographic
symmetry are located along the x-, y- and z-directions. The peaks representing the non-
crystallographic symmetry elements are highlighted and labelled by the respective subunit
identifiers. Blue circles show the twofold axes relating the two monomers within one LiGLO1 dimer
and red circles the twofold axes relating the monomers of different dimers to each other. In addition,
the position of the non-crystallographic threefold axis is indicated by a green triangle (Note: The peak
corresponding to the green triangle does of course not appear on the κ = 180° section depicted here,
but on the κ = 120° section instead). The three dimers of LiGLO1 in the asymmetric unit assemble to a
hexamer of approximate D3-symmetry. This figure was produced using the program MOLREP
(Vagin & Teplyakov, 1997).
The structure of LiGLO1 was solved by molecular replacement using the related structure of
glyoxalase I from L. major (PDB entry 2C21 (Ariza et al. 2006)) and the lower resolution data set
collected from the initial form 2 crystals. After placing three dimers in the asymmetric unit, the
correlation coefficient was 64.2 % and the R-factor to 3.5 Å resolution 39.6 %. This essentially confirms
the correctness of the structure solution. In retrospect, the self-rotation function (Figure II.2.) can be
examined and interpreted using the known structure as a guide. The three dimers of LiGLO1 arrange
themselves in a pseudo-D3-symmetric arrangement, with the threefold axis only about 14º away from
the crystallographic z-axis and all twofold axes close to the xy-plane. A very similar arrangement has
also been observed in the structure of LmGLO1 (Ariza et al. 2005).
CChhaapptteerr IIII
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44..44.. OOvveerraallll ddeessccrriippttiioonn ooff tthhee LLiiGGLLOO11 ccrryyssttaall ssttrruuccttuurree
The asymmetric unit of the orthorhombic crystal form contains six polypeptide
chains, which form three dimers named AB, CD and EF. The LiGLO1 structure was refined
against data extending to a resolution of 2.1 Å in space group P21212, resulting in an R factor
of 18.0 %, and a corresponding Rfree (Brunger 1992) of 22.0 %. The final polypeptide chains
have between 139 and 141 residues. Each protein dimer has two metal ions and two MPD (2-
methyl-2,4-pentanediol) molecules. A total of 285 water molecules were found in the
structure. The average temperature factor of the protein is 40.6 Å2. The rmsd deviation
values from ideal bond lengths and bond angles were 0.015 Å and 1.7°, respectively, and no
outlier residues were found in the Ramachandran plot (Kleywegt et al. 1996). Based on these
quality criteria, the structure can be considered well refined and of high quality.
LiGLO1 is a 141 amino acid metalloprotein arranged in dimers, as referred. Like the
other previously described glyoxalase I structures (Cameron et al. 1997; He et al. 2000; Ariza
et al. 2006), LiGLO1 is arranged in two βαβββ domains. An eight-stranded β-sheet with
pseudo-twofold symmetry emerges due to the dimer formed by the interaction of the first
domain of one monomer with the second domain of the other (Figure II.3.). Each dimer has
two metal binding sites named AB1, AB2, CD1, CD2, EF1 and EF2, respectively. The metal
binding sites are located in the interface of two monomers building up the dimer. The side
chains of His8, Glu59, His77' and Glu120' (where prime “ ' “ indicated the second monomer)
are responsible for coordination of the metal ion. Two sodium ions are located at the
interface between the dimers AB and CD, as well as between CD and EF (Figure II.3.). Dimer
AB was used for all subsequent structural interpretation.
CChhaapptteerr IIII
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Figure II.3. LiGLO1 structure. (a) Content of the asymmetric unit. Chain A (blue), B (yellow), C
(green), D (pink), E (gold) and F (red) are shown. Water molecules (red) interacting with zinc ions
(light blue), sodium ions (yellow) and MPD molecules are depicted. (b) Dimer AB. Chain A (blue), B
(yellow), water molecules (red) interacting with zinc ions (light blue), sodium ions (yellow) and MPD
molecules are depicted. (c) Metal-binding site. Zinc ion (light blue) is coordinated by His8 and Glu59
from chain A (blue), His77’ and Glu120’ from chain B (yellow) and a water molecule (red). Distances
are indicated. The figure was prepared using UCSF Chimera (Pettersen et al. 2004).
LiGLO1 is structurally very similar to Ni2+-dependent LmGLO1 (Ariza et al. 2006)
(rmsd of 0.27 Å superposing dimer AB, based on 273 Cα atoms), differing in only three
residues located at the protein surface: Gln39, Ala50 and Met126 of LiGLO1 corresponding
to Glu39, Gly50 and Thr126 in LmGLO1. Moreover, LiGLO1 is more similar to the
prokaryotic Ni2+-dependent EcGLO1 (He et al. 2000) (rmsd of 1.02 Å, superposing dimer AB,
based on 234 Cα atoms) than to the Zn2+-dependent HsGLO1 (Cameron et al. 1997) (rmsd of
1.48 Å, superposing dimer AB, based on 230 Cα atoms), as also observed for the LmGLO1
enzyme (Ariza et al. 2006). However, significant differences are observed between LiGLO1
CChhaapptteerr IIII
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and both EcGLO1 and HsGLO1. The loop between strands 6 and 7 is seven and five residues
shorter relatively to EcGLO1 and HsGLO1, respectively, and the 15-residue C-terminal helix
is absent from EcGLO1 and shorter and in a different orientation in HsGLO1.
44..55.. AAccttiivvee SSiittee
The active site of glyoxalase I consists of a highly conserved catalytic metal centre
and γ-glutamate-binding residues, and the methylglyoxal pocket and the glycylcarboxylate-
or amide-binding residues, both showing significant differences across structures.
Although we tried to solve a structure of LiGLO1 in complex with the hemithioacetal
derived from glutathione, trypanothione and glutathionil-spermidine, by soaking crystals
with this substrates, a MPD molecule was always present in the active site. This compound
is essential in the crystallization condition at a concentration as high as 3M and co-
crystallised with the protein. Similarly to what was described for LmGLO1 (Ariza et al. 2006),
this MPD molecule interacts with Try35, a residue absent from EcGLO1 and HsGLO1.
A divalent metal is required to polarise both oxygen atoms of the methylglyoxal
moiety, facilitating the rearrangement of the substrate to D-lactate (Bailey et al. 1994; Himo &
Siegbahn 2001). In the LiGLO1 structure, Zn2+ is coordinated by residues His8 and Glu59,
from one monomer, and His77 and Glu120, from another monomer, and a water molecule
(Figure II.3.). The distances between zinc ion and the coordinating atoms (> 2.13 Å) are
longer than typical for this ion (approximately 2.00 Å (Harding 1999)), suggesting the metal-
binding sites are not fully occupied, similarly to LmGLO1 (Ariza et al. 2006). This was based
on the EcGLO1 structure (He et al. 2000), the only one to reveal fully occupied metal-binding
sites. However, the distances in EcGLO1 are smaller than the ones found for LmGLO1,
showing the different metal content of both enzymes, since nickel-ion distances are typically
longer (approximately 2.06 Å (Harding 1999)) than the zinc-ion ones. The metal composition
of LiGLO1 will be described in detail in the next section of this Chapter.
In spite of the metal difference observed, and although LiGLO1 active site surface
(Figure II.4.) is slightly different relatively to EcGLO1 (He et al. 2000) and HsGLO1 (Cameron
et al. 1997), it is very similar to LmGLO1 (Ariza et al. 2006). Also, the residues binding the γ-
glutamate moiety of the substrate, Asn63 and Arg12, are conserved between the GLO1
analysed structures.
CChhaapptteerr IIII
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LiGLO1 structure, apart from having diverse substrate specificity from HsGLO1,
shows significant structural differences towards the human counterpart, including its less
accessible active site mainly due to interactions between β6 and β7 and the loop between β2’
and β3’.
Figure II.4. Molecular surface of LiGLO1 dimer AB (chain A in blue; chain B in yellow). Coloured
residues are: zinc ion coordinating (orange), MPD binding (red), conserved on γ-glutamyl moiety
binding (light green) and differing from LmGLO1 (pink) are coloured. Zinc ion is in light blue. The
figure was prepared using UCSF Chimera (Pettersen et al. 2004).
44..66.. MMeettaall ccoommppoossiittiioonn
To investigate the LiGLO1 metal-content, Inductively-Coupled Plasma (ICP), an atomic
emission spectrometry method, was used (Faires 1993). LiGLO1 purified on Co-NTA was analysed
targeting zinc, nickel and cobalt. Although the protein was purified using a cobalt-affinity column,
the analysis revealed that of the three metals analysed LiGLO1 contained only zinc.
In order to further analyse which metal-ion clearly occupies the metal binding site of
the active site, we exploited X-ray anomalous scattering properties of the transition metals.
Four 0.5 mm long and 0.15 mm thick crystals, two from each Co-NTA and Ni-NTA
purification batches (referred to as crystal 1 and crystal 2 from each purification batch),
respectively, were used for these measurements without the need for cryoprotectant. X-ray
fluorescence spectra were collected for crystals at the EMBL Hamburg X12 beamline at
CChhaapptteerr IIII
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DORIS storage ring, DESY, Hamburg. Incident X-rays of 9950 eV, 8600 eV, 8000 eV energy
were chosen to excite the elements zinc, nickel and cobalt, respectively, accordingly to the
metals absorption edges (Figure II.5.). The fluorescence spectrum (Figure II.6a.) clearly
shows zinc as the main metal bound to LiGLO1, being nickel and cobalt present in very
small amount. Also detected in low quantities are copper and manganese, commonly
detected when zinc is present; and iron, attributed to the pin used for measurement, and not
considered in the subsequent analysis (Figure II.6b.).
Figure II.5. Theoretical absorption edges of zinc, cobalt and nickel. The respective anomalous
components ∆f ” are shown as a function of incident X-ray energy. The energy values used for data
collection, indicated by black arrows, were taken from the site of the Biomolecular Structure Center at
the University of Washington, Seattle, USA.
(http://skuld.bmsc.washington.edu/scatter/AS_periodic.html).
CChhaapptteerr IIII
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Figure II.6. X-ray fluorescence spectra of (a) LiGLO1 crystal and (b) loop containing only reservoir
solution. In (a) zinc (Zn) corresponds to the most intense peak, being nickel (Ni) and cobalt (Co) peaks
very weak. Copper (Cu) and manganese (Mn) weak peaks are also visible. These metals are usually
detected when zinc is present. In (b) only the peak for iron (Fe) is visible, being this metal attributed
to the pin used for measurement.
For the crystal 1 (from each purification batch), data were collected stepwise increasing
the energy, while for crystal 2 (from each purification batch) data were collected by
decreasing the energy. In both cases the data sets were collected starting at the same phi
angle and the detector distance was adjusted, to obtain similar resolution limits for all data
sets collected. Crystal 1 and crystal 2 from the Co-NTA batch diffracted to 2.17 Å (Table II.
2.)., while crystal 1 and crystal 2 from the Ni-NTA batch diffracted to 2.18 Å (Table II.3.).
CChhaapptteerr IIII
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Table II.2. Data collection and processing from LiGLO1 crystal purified on Co-NTA. Values in parentheses are for the highest resolution shell.
LiGLO1 purified on Co-NTA Crystal 1 Crystal 2
Energy (eV) 9950 8600 8000 7500 7500 8000 8600 9950 No. of crystals 1 1 Wavelength (Å) 1.24603 1.44163 1.54975 1.65307 1.65307 1.54975 1.44163 1.24603
Crystal-to-detector distance (mm) 180 150 133 120 120 133 150 180 Rotation range per image (deg) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Total rotation range (deg) 180 180 180 180 180 180 180 180 Space group P21212 P21212 Cell dimensions a,b,c (Å) 129.53,
147.38, 50.24
129.54 147.39 50.24
129.58, 147.44, 50.26
129.54, 147.39, 50.24
129.49, 147.41, 50.25
129.54, 147.46, 50.27
129.52, 147.43, 50.26
129.52, 147.42, 50.26
Resolution (Å) 50 - 2.25 (2.30-2.25)
50 - 2.25 (2.30-2.25)
50 - 2.25 (2.30-2.25)
50 - 2.25 (2.30-2.25)
50 - 2.20 (2.25-2.20)
50 - 2.20 (2.25-2.20)
50 - 2.20 (2.25-2.20)
50 - 2.20 (2.25-2.20)
Completeness (%) 99.4 (94.4) 99.7 (98.4) 99.5 (97.0) 99.5 (96.7) 99.5 (96.2) 99.4 (95.2) 99.5 (95.7) 99.5 (96.2) Redundancy 3.8 (3.3) 3.8 (3.2) 3.8 (3.5) 3.8 (3.6) 3.7 (2.9) 3.7 (2.9) 3.7 (2.7) 3.8 (2.9) Mosaicity (deg) 0.167 0.149 0.150 0.144 0.134 0.132 0.131 0.132 I / σ(I) 11.2 (2.9) 19.1 (2.9) 17.6 (2.7) 15.6 (2.1) 14.0 (2.2) 14.4 (2.2) 14.4 (2.1) 15.2 (2.6) Rmerge (%) 7.8 (39.0) 4.7 (39.2) 5.2 (45.3) 6.0 (59.1) 6.0 (48.6) 5.9 (49.4) 5.9 (47.1) 5.6 (40.8)
Rr.i.m. (%) 9.1 (46.3) 5.5 (46.9) 6.0 (53.3) 6.9 (69.3) 7.1 (59.0) 6.9 (59.3) 6.8 (57.8) 6.6 (49.6) Rp.i.m. (%) 4.7 (25.5) 2.8 (26.2) 3.1 (28.5) 3.5 (36.5) 3.7 (34.6) 3.6 (34.8) 3.5 (35.2) 3.4 (29.1) Anomalous Correlation (%) 13 (1) 6(2) 6 (3) 7 (3) 7 (1) 6 (3) 6 (5) 20 (3) B-factor from Wilson plot (Å) 42.21 42.67 43.24 43.95 40.66 41.14 41.62 41.51 Optical resolution (Å) 1.75 1.76 1.76 1.76 1.73 1.74 1.74 1.74
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Table II.3. Data collection and processing from LiGLO1 crystal purified on Ni-NTA. Values in parentheses are for the highest resolution shell.
LiGLO1 purified on Ni-NTA Crystal 1 Crystal 2
Energy (eV) 9950 8600 8000 7500 7500 8000 8600 9950 No. of crystals 1 1 Wavelength (Å) 1.24603 1.44163 1.54975 1.65307 1.65307 1.54975 1.44163 1.24603
Crystal-to-detector distance (mm) 180 150 133 120 120 133 150 180 Rotation range per image (deg) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Total rotation range (deg) 180 180 180 180 180 180 180 180 Space group P21212 P21212 Cell dimensions a,b,c (Å) 129.16
147.48 50.30
129.17 147.48 50.30
129.18 147.45 50.29
129.18 147.45 50.28
129.39 147.63 50.30
129.23 147.46 50.24
129.25 147.47 50.24
129.26 147.47 50.24
Resolution (Å) 50-2.17 (2.22-2.17)
50-2.17 (2.22-2.17)
50-2.17 (2.22-2.17)
50-2.17 (2.22-2.17)
50 – 2.18 (2.24-2.18)
50 – 2.18 (2.24-2.18)
50 – 2.18 (2.24-2.18)
50 – 2.18 (2.24-2.18)
Completeness (%) 99.8 (99.1) 99.7 (97.6) 99.7 (98.6) 99.7 (98.5) 99.7 (97.8) 99.6 (96.9) 99.7 (97.7) 99.6 (96.7) Redundancy 3.7 (2.7) 3.7 (2.4) 3.7 (2.6) 3.7 (2.6) 3.7 (2.7) 3.7 (2.7) 3.7 (2.5) 3.7 (2.8) Mosaicity (deg) 0.101 0.109 0.116 0.122 0.114 0.118 0.115 0.107 I / σ(I) 27.9 (5.8) 25.6 (4.2) 24.8 (3.5) 22.4 (2.1) 19.9 (2.0) 19.9 (2.1) 20.4 (2.4) 20.0 (2.9) Rmerge (%) 3.3 (17.2) 3.5 (22.8) 3.6 (29.4) 4.1 (48.8) 5.0 (50.9) 5.0 (50.8) 4.8 (42.1) 4.9 (36.9)
Rr.i.m. (%) Rp.i.m. (%) Anomalous Correlation (%) 25 (4) 12 (2) 8 (5) 11 (3) 10 (7) 7 (6) 9 (3) 15 (4) B-factor from Wilson plot (Å) 31.9 34.8 36.7 40.2 38.0 38.7 37.8 36.6 Optical resolution (Å) 1.67 1.68 1.68 1.68 1.69 1.69 1.69 1.69
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Table II.4. Peaklist of LiGLO1 purified on Co-NTA and Ni-NTA, respectively. Given is the electron density at the metal site (AB1, AB2, CD1, CD2, EF1 and EF2,
respectively) and at the strongest sulfur peak from each dimer. The energy values used for data collection are given in eV.
LiGLO1 purified on Co-NTA LiGLO1 purified on Ni-NTA
Crystal 1 Crystal 2 Crystal 1 Crystal 2
Energy (eV) 9950 8600 8000 7500 7500 8000 8600 9950 9950 8600 8000 7500 7500 8000 8600 9950
Electron density at metal sites (σ units):
AB1 25.6 6.2 5.6 7.4 8.9 6.8 5.7 31.7 34.1 8.7 8.4 8.7 6.0 7.4 6.9 25.9
AB2 19.6 6.7 5.6 5.9 6.2 6.5 4.6 23.7 32.1 16.0 7.4 7.4 8.4 6.7 12.4 24.8
CD1 20.1 7.7 6.3 6.7 6.2 4.9 7.4 26.1 34.4 22.6 9.5 7.5 7.0 7.2 15.3 24.5
CD2 26.0 7.7 6.7 4.4 6.8 6.9 7.1 33.9 34.5 12.5 8.4 8.7 7.6 7.0 8.0 26.8
EF1 16.3 5.3 6.2 5.1 6.1 4.7 5.9 21.7 27.9 17.5 7.4 7.3 7.1 6.9 11.4 21.1
EF2 13.0 6.4 5.7 4.3 5.0 4.0 4.9 17.5 22.0 18.6 6.8 4.9 5.5 7.7 13.3 17.4
Electron density at strongest sulfur sites (σ units):
AB 3.9 6.5 5.5 6.4 6.7 6.7 5.8 4.5 7.4 10.0 10.1 10.5 8.2 7.3 6.9 6.1
M29B M135A M29B M10B M135A M29B M29B M10A M29B M29B M10A M10A M10A M29A M10A M29B
CD 4.6 7.6 7.7 5.9 8.0 7.1 6.4 4.7 7.1 10.3 11.1 11.0 10.3 8.4 8.6 5.5
M10C M29C M29C M29C M135C M10D M29C M10C M29C M10D M29C M29C M29C M29C M10D M29C
EF 4.2 5.4 6.2 5.2 4.9 6.6 5.4 4.0 5.8 7.3 8.5 8.7 7.1 6.4 6.4 4.8
M127E M10F M10E M29E M29E M10E M10E M128F M10E M10E M10E M10F M10E M10E M10F M10E
CChhaapptteerr IIII
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The final coordinates of the native structure were used. For the analysis of the anomalous
scattering data, only one round of refinement in Refmac5 (Murshudov et al. 1997) was carried out.
The resulting models and the anomalous differences at each wavelength were used to calculate an
anomalous difference map and to assign the peaks to surrounding atoms of the native coordinates
using programs from the CCP4 suite (FFT, MAPMASK,PEAKMAX and WATPEAK) (Bailey 1994).
The sigma levels of the anomalous difference peaks at the two metal binding sites of each of the
three dimers in the asymmetric unit as well as the strongest anomalous difference peak of one
sulfur from each dimer, are given in Table II.4.
The occupancies of metal binding sites scaled to the intrinsic anomalous difference of sulfur
are presented in Table II.5. Results suggest that zinc is the main binder at the metal binding site, in
agreement with the information from the ICP and the fluorescence based metal analysis, showing
67.5 % and 73.1 % occupancy for Co-NTA batch crystals 1 and 2, respectively, and 45.5 % and 38.8 %
for Ni-NTA batch crystals 1 and 2, respectively. For all binding sites, the cobalt amount in Ni-NTA
crystals is significantly lower than in Co-NTA ones. Nickel is present in Co-NTA crystals in small
amounts (up to 6.8 %), while in Ni-NTA crystals its quantity is increased of a maximum of around
20 %, showing that the enzyme metal-content depends to some extent on the affinity column metal
used in the purification procedure, but not enough to completely shift it. These results confirm that
zinc is the physiological LiGLO1 preferential metal. No significant difference was observed when
collecting data in opposite directions of energy for crystals 1 (from higher energy to the lowest) and
2 (from lower energy to the highest) (Table II.6), showing that the crystal metal-content is
independent from data collection strategy. Radiation damage affects the overall crystal diffraction,
as well as the sulphur peaks, used as a scaling reference for the metal peaks. Data collection in both
directions assures there is no associated error to the calculated values.
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Table II.5. Occupancies of metal binding sites scaled to sulfur. Metal binding sites are named AB1, AB2, CD1,
CD2, EF1 and EF2.
Binding site LiGLO1 purified on Co-NTA LiGLO1 purified on Ni-NTA
Crystal 1 Crystal 2 Crystal 1 Crystal 2 AB1 Zn (%) Ni (%) Co (%)
67.5 0.0 2.3
73.1 0.0 1.5
45.0 0.6 4.2
38.8 0.0 8.1
AB2 Zn (%) Ni (%) Co (%)
48.4 0.2 6.4
52.7 0.0 3.7
32.6 13.2 3.2
27.9 13.3 6.9
CD1 Zn (%) Ni (%) Co (%)
41.1 2.9 4.3
52.3 6.8 0.0
31.1 20.4 4.3
31.9 14.0 5.1
CD2 Zn (%) Ni (%) Co (%)
55.0 2.2 2.6
72.1 2.2 0.9
43.7 7.0 2.3
47.3 1.5 3.6
EF1 Zn (%) Ni (%) Co (%)
35.2 0.0 8.5
52.5 5.8 0.2
28.8 23.2 4.6
32.1 10.6 8.9
EF2 Zn (%) Ni (%) Co (%)
23.4 4.0 9.1
42.1 4.6 0.7
14.3 26.6 5.8
18.8 13.2 13.0
CChhaapptteerr IIII
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44..77.. LLiiGGLLOO11 KKiinneettiicc AAnnaallyyssiiss
Recombinant LiGLO1 activity was assayed for the hemithioacetals formed between
methylglyoxal and either glutathione or trypanothione. A Km for trypanothione-derived HTA of
0.27 mM and a Km for glutathione-derived HTA of 0.97 mM were determined (Table II.6.). The
obtained Km values are consistent with our previous results using L. infantum extracts (Km of 0.24
mM for trypanothione-derived HTA and Km of 1.85 mM for glutathione-derived HTA) (Sousa Silva
et al. 2005). The kcat/Km values of 6.60x103 mM-1s-1 and 0.72x103 mM-1.s-1 for trypanothione- and
glutathione-derived HTA, respectively, indicate rapid turnover of these substrates.
Table II.6. Kinetic parameters of recombinant LiGLO1 and comparison to GLO1 from L. infantum total protein
extracts and to recombinant GLO1 from L. donovani, L. major and human.
Hemithioacetal
Substrate Km (mM)
kcat (s-1)
kcat/Km (mM-1.s-1)
LiGLO1 recombinant
T(SH)2-derived 0.270 1800 6.60x103
GSH-derived 0.970 698 0.72x103 LiGLO1
in total protein extractsa T(SH)2-derived 0.240 - - GSH-derived 1.850 - -
L. major GLO1b T(SH)2-derived 0.032 800 25x103
GSH-derived >1900 - 0.13x103
L. donovani GLO1c T(SH)2-derived 0.028 - - Human GLO1d GSH-derived 0.071 - 0.22x103
aValues from Sousa Silva et al. 2005; bValues from Vickers et al. 2004; cValues from Padmanabhan et al. 2005; dValues from Ridderstrom & Mannervik 1996.
Our results confirm that LiGLO1 shows affinity towards both trypanothione and
glutathione-derived substrates, although it preferentially reacts with the trypanothione-derived
hemithioacetal. LiGLO1 shows a greater turnover for the trypanothione hemithioacetal (kcat of
1.8x103 s-1) than LiGLO2 for S-D-lactoyltrypanothione (kcat of 3.52 s-1, see chapter III of this thesis),
consistent with previous obtained values for both enzymes from the human glyoxalase system (Rae
et al. 1990).
Together with the solved structure and the metal and kinetic analysis of LiGLO2 later
described on this work, the availability of the LiGLO1 structure, kinetics and metal-content,
CChhaapptteerr IIII
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reported here, assigns a better understanding of the glyoxalase pathway in trypanosomatids,
revealing the structural features concerning its exclusive substrate specificity and inducing its
uniqueness.
44..88.. LLiiGGLLOO11 eevvoolluuttiivvee ccoonnssiiddeerraattiioonnss
The LiGLO1 detailed metal analysis comes as one more clue to the much discussed metal-
content subject. In general, GLO1 from eukaryotes contain zinc in their active site, differing from
nickel-dependent GLO1 from prokaryotes. So far, there is only one example in literature where an
eukaryotic GLO1, LmGLO1 (Ariza et al. 2006), uses nickel instead of zinc. In our study, LiGLO1
contains zinc in the active site, despite the affinity metal used in the purification process. This
indicates an earlier evolutive divergence of LiGLO1 from the other eukaryotes than its structurally
very close L. major homologue enzyme. Hence, the predicted similarity by sequence homology is
relinquished, as a critical difference is established between these two structurally similar enzymes
(97 % identity) and enhancing the importance of studying LiGLO1.
55.. AAcckknnoowwlleeddggmmeennttss
The authors acknowledge the European Synchrotron Research Facility (ESRF, Grenoble,
France) and the EMBL Hamburg at the Deutsches Elektronen-Synchrotron (DESY, Hamburg,
Germany) for access to synchrotron beamtime. The work was supported by projects
POCTI/ESP/48272/2002 and POCI/QUI/62027/2004 (Fundação para a Ciência e Tecnologia,
Portugal). LB was supported by the doctoral grant SFRH/BD/28691/2006 from the Fundação para
a Ciência e Tecnologia (Portugal) and by an EMBO short-term fellowship ASTF 318-2008.
Trincão, J.‡, Sousa Silva, M.‡, Barata, L., Bonifácio, C., Carvalho, S., Tomás, A. M., Ferreira, A., Cordeiro, C.,
Ponces Freire, A., Romão, M. J. (2006) Purification, Crystallization and Preliminary X-ray Diffraction Analysis
of the Glyoxalase II from Leishmania infantum, Acta Crystallographica Section F 62, 805-807. (‡ both authors
contributed equally for the work)
Sousa Silva, M. ‡, Barata, L.‡, Ferreira, A. , Romão, S., Tomás, A. M., Ponces Freire, A. and Cordeiro, C.
(2008) Catalysis and Structural Properties of Leishmania infantum Glyoxalase II: Trypanothione Specificity and
Phylogeny, Biochemistry 47, 195-204. (‡ both authors contributed equally for the work)
Manuscript on mutated LiGLO2 in preparation.
CChhaapptteerr IIIIII GGllyyooxxaallaassee IIII ffrroomm LLeeiisshhmmaanniiaa iinnffaannttuumm
CChhaapptteerr IIIIII
5599
11.. SSuummmmaarryy
The glyoxalase pathway catalyses the formation of D-lactate from methylglyoxal, a toxic by-
product of glycolysis. In trypanosomatids, trypanothione replaces glutathione in this pathway,
making it a potential drug target, since its selective inhibition might increase methylglyoxal
concentration in the parasites. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I
and glyoxalase II, the latter shows absolute specificity towards trypanothione thiolester, making
this enzyme an outstanding model to understand the molecular basis of trypanothione binding.
Cloned glyoxalase II from Leishmania infantum was over-expressed in E. coli, purified and
crystallised. Crystals belong to space group C2221 (a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyond
2.15 Å using synchrotron radiation Two glyoxalase II structures were solved by Molecular
Replacement using the human glyoxalase II structure as a search model. One with a bound
spermidine molecule (1.8 Å) and the other with D-lactate at the active site (1.9 Å). The second
structure was obtained by crystal soaking with the enzyme substrate S-D-lactoyltrypanothione.
Overall structure of L. infantum is very similar to its human counterpart, with important differences
at the substrate binding site. The crystal structure of L. infantum glyoxalase II (LiGLO2) is the first
structure of this enzyme from trypanosomatids. The differential specificity of glyoxalase II towards
glutathione and trypanothione moieties was revealed by the differential substrate binding.
Evolutionary analysis shows that trypanosomatid glyoxalases II diverged early from eukaryotic
enzymes, being unrelated to prokaryotic proteins.
Native LiGLO2 revealed crucial differences relatively to the human homologue, namely at
the substrate-binding residues. Here we report the identification of the essential residues for S-D-
lactoyltrypanothione binding, Tyr291 and Cys294, and their mutation to the corresponding residues
in the human enzyme interacting with S-D-lactoylglutathione, Arg249 and Lys252, respectively.
LiGLO2 substrate-specificity changed and the mutated enzyme was able to hydrolyse the
glutathione-derived thiolester. New kinetic parameters for both glutathione- and trypanothione-
derived substrates were determined and metal analysis was undertaken. This study allowed a
detailed analysis of a model trypanosomatid glyoxalase II concerning the essential residues for
substrate specificity.
CChhaapptteerr IIIIII
6600
22.. IInnttrroodduuccttiioonn
The trypanosomatids are the etiological agents of several human and animal diseases,
widespread in the third world and in the Mediterranean basin. Emergent target populations for
leishmaniasis are HIV patients and people following immunosupressor therapies. No curative
drugs or vaccines currently exist and actual therapeutic approaches are becoming limited given
their undesirable side effects and the evolution of resistant forms of trypanosomatids. Novel and
effective therapeutic approaches are much needed and these must rely on unique physiological and
biochemical properties of trypanosomatids. These intracellular eukaryotic parasites have a complex
life cycle, requiring an insect vector for transmission to a mammalian host. Unique cytological
features are the presence of a single large mitochondrion containing the kinetoplast DNA and a
singular compartment, the glycosome, where the major part of glycolysis takes place (Michels et al.
2000; Hannaert et al. 2003). However, the most remarkable biochemical characteristic of
trypanosomatids is the functional replacement of glutathione by trypanothione (N1,N8-
bis(glutathionyl)-spermidine), a spermidine-glutathione conjugate (Muller et al. 2003; Flohe et al.
1999). This thiol fulfils the role of glutathione in the detoxification of metals, drugs, hydroperoxides,
peroxynitrite, and ketoaldehydes (Krauth-Siegel et al. 2005). Glutathione-dependent enzymes in
trypanosomatids are replaced by functionally analogue enzymes that specifically use
trypanothione. For instance, trypanothione reductase replaces glutathione reductase, while
thioredoxin dependent processes are replaced by the tryparedoxin system (Flohe 1999). Not
surprisingly, trypanothione is essential and therefore, targeting its biosynthesis or trypanothione
dependent enzymes with inhibitors will provide a synergistic effect with deleterious consequences
to the parasite, while leaving unaffected the host glutathione dependent processes. To achieve this
goal, the focus should be placed on researching the molecular basis of trypanothione specificity. An
ideal model is glyoxalase II, an enzyme from the glyoxalase system. Through the sequential action
of glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) and glyoxalase II (hydroxyacyl glutathione
hydrolase, EC 3.1.2.6), D-lactate is produced from methylglyoxal using glutathione in every living
cell (Thornalley 1990), except in trypanosomatids where the thiol is trypanothione (Vickers et al.
2004; Irsch & Krauth-Siegel 2004; Sousa Silva et al. 2005). Glyoxalase I catalyses the formation of
lactoyltrypanothione from the hemithioacetal formed non-enzymatically from methylglyoxal and
trypanothione, while glyoxalase II hydrolyses this thiolester forming D-lactate, regenerating
trypanothione. Both enzymes catalyse virtually irreversible reactions and glyoxalase II shows
CChhaapptteerr IIIIII
6611
absolute specificity towards the trypanothione moiety in lactoyltrypanothione (Irsch & Krauth-
Siegel 2004; Sousa Silva et al. 2005).
In the present study, recombinant L. infantum GLO2 (LiGLO2) was cloned, purified,
crystallised and kinetically characterised. Its crystal structure was solved by molecular replacement,
using the crystal structure of the glutathione-dependent human glyoxalase II (Cameron et al. 1999).
LiGLO2 is a 295 amino acid monomeric metalloprotein, arranged in two domains. Its structure is
similar to the whole structure of the human homologue (PDB entry 1QH3 (Cameron et al.
1999).Several highly conserved residues are shared by the trypanosomatid and human glyoxalases
II at the active site. This is not surprising since the catalysed reaction is chemically identical and
both are metallo-β-lactamases containing zinc or iron at the active site. However, some residues are
unique to the trypanosomatid enzyme and their location close to the active site explains the
differential substrate specificity. Trypanosomatids lack the Lys143, Arg249, and Lys252 responsible
for glutathione binding in human GLO2, while LiGLO2 has Ile171 (absent from the human enzyme)
and both Phe219 and Phe266 strategically positioned to bind the spermidine moiety of the thiolester
(Sousa Silva et al. 2008) and absent from the binding site in the human structure. Two of the
residues identified as determinant for glutathione binding, Arg249 and Lys252 in the human
homologue enzyme, correspond to the L. infantum substrate binding site residues Tyr291 and
Cys294, respectively. In this work, we also report the double mutation, Y291R and C294K in
LiGLO2. The mutant LiGLO2 was subsequently cloned, expressed, purified and kinetically
characterised.
This is the first report of the glyoxalase II structure from a trypanosomatid. The presence of
an extra density compatible with a spermidine molecule bound close to the active site and the
characterization of the mutated form of the enzyme allowed us to understand why trypanosomatid
glyoxalases II are specific towards trypanothione-derived substrates, approaching a full insight into
the glyoxalase pathway along evolution.
CChhaapptteerr IIIIII
6622
33.. MMaatteerriiaall aanndd MMeetthhooddss
33..11.. LLiiGGLLOO22 cclloonniinngg:: nnaattiivvee aanndd mmuuttaanntt ffoorrmmss
The native (LiGLO2) and the mutant form (LiGLO2m) of glyoxalase II gene were amplified
from L. infantum (clone MHOM/MA67ITMAP263) genomic DNA by PCR with specific primers.
The sense primer (5’-ccgcgcacatatgcgcaactactgcac-3’) contained the Nde I site (underlined) and the
start ATG (italic). The anti-sense primer (5’-caccgctcgagtcagtcgcaggcgttgt-3’ for the native form, and
5’-caccgctcgagtcagtccttggcgttgcgaaggtacatcatcagcgc-3’ for the mutant form, with the altered
nucleotides indicated in bold) contained the Xho I site (underlined) immediately after the stop
codon. The genes were amplified using the PWO polymerase (GibcoBRL), as follows: 94 °C for 2
min, 48 ºC for 1 min, 72 °C for 1 min – 2 cycles; 94 °C for 45 sec, 62 °C for 1 min, 72 °C for 1 min – 30
cycles; 72 °C for 10 min. Both genes were cloned into the prokaryotic expression vector pET-28a
(Novagen), using the referred restriction sites, and E. coli BL21-codon plus (Stratagene) were
transformed with the pET-28a/His6-LiGLO2 and pET-28a/His6-LiGLO2m plasmids. Sequencing of
both open reading frames was carried out prior to protein expression.
33..22.. PPrrootteeiinn eexxpprreessssiioonn aanndd ppuurriiffiiccaattiioonn
For over-expression of His6-LiGLO2 and His6-LiGLO2m (fusion proteins, with an N-terminal
tail of six histidines and a thrombin cleavage site; total tag sequence including linker and cleavage
site (underlined) is MGSSHHHHHHSSGLVPRGSH), transformants were grown in 3 L of LB
medium containing 50 µg.ml-1 kanamycin and 34 µg.ml-1 chloramphenicol, at 37 ºC. When the
culture reached an OD600nm of 0.6, expression was induced with 0.2 mM isopropyl-β-D-
thiogalactopyranoside (IPTG), for 3 hours at 37 °C. After induction, cells were harvested by
centrifugation at 7800 g for 30 min at 4 °C, and the cell pellets were frozen at -20 °C.
LiGLO2 cell pellet was resuspended in 120 ml 500 mM NaCl, 20 mM Tris-HCl pH 7.6, lysed
by sonication and the cell debris removed by centrifugation at 10.000 ×g for 30 min at 4°C. The
fusion protein was purified at 4 °C by chromatography on a His Bind resin (Novagen) column (XK
26/20; Amersham Pharmacia Biotech, Uppsala, Sweden). His-LiGLO2 was eluted with an imidazole
gradient from 5 mM to 1 M at a flow rate of 2.5 ml.min-1. Fractions confirmed to contain LiGLO2 by
CChhaapptteerr IIIIII
6633
SDS-PAGE were pooled, applied to PD-10 columns (Amersham Biosciences) and eluted with 1x PBS
pH 7.4. His6-LiGLO2 was concentrated using Amicon® Ultra-4 filters (10,000 NMWL, Millipore
Corporation) and further purified by FPLC, with a LKB 2050 pump (LKB-Bromma) using a
SuperdexTM 75 10/300 GL column (Amersham Biosciences), and a Jasco UV-2075 Plus detector at
280 nm, with a flow of 0.4 mL.min-1, in 150 mM NaCl and 20 mM KH2PO4. Protein concentration
was determined using the Bio-Rad protein assay dye reagent (Bio-Rad).
Ressuspension of LiGLO2m required an optimised buffer. The cell pellet of LiGLO2m was
suspended in 10 ml of buffer A (30 mM Tris pH 8.5, 100 mM NaCl, 5mM β-mercaptoethanol, 5mM
MgCl2, all chemicals were obtained from Carl Roth) per g of wet cell pellet. Buffer A was optimised
by ligand-assisted monitoring of thermal protein unfolding in a ThermoFluor (Biorad) experiment
(Ericsson et al. 2006), which showed to be crucial for purification improvement. 5 µl 50 × Sypro
Orange (fluorophore; Invitrogen), 5 µl of 1 mg/ml protein and 40 µl of each buffer were mixed in a
96-well thin-wall PCR plate (Bio-Rad). The plate was heated (4 to 95 °C) in increments of 0.5 °C and
fluorescence detected in a ThermoFluor ICycler IQ thermal cycler (Bio-Rad). Two screens were
performed: a pH versus NaCl concentration screen and an additives screen using 50mM Tris-HCl
pH 8.5 as the basis buffer. After ressuspension, cells were lysed by sonication. The soluble fraction
obtained by centrifugation at 38000 g for 40 min at 4°C was filtered through a 0.22 µm membrane,
before being applied to a cobalt-charged affinity column for purification at room temperature. The
fusion protein was eluted in buffer A supplemented with an optimised concentration of 300 mM
imidazole, and then directly applied to a gel filtration column (Superdex S75 16/60, GE Healthcare)
equilibrated with buffer A for further purification. The protein was concentrated using a Vivaspin15
(molecular weight cutoff 10000) concentrator (Sartorius Stedim Biotech, Göttingen, Germany).
Protein concentration was determined using a Peqlab Biotechnologie GmbH NanoDrop® ND-1000
Spectrometer at 280 nm, assuming an estimated extinction coefficient of 22015 M-1 cm-1 for the
mutant form of LiGLO2, as calculated by ProtParam tool (Gasteiger et al., 2005) for a protein
molecular weight of 33.4 kDa.
The purity of His6-LiGLO2 and His6-LiGLO2m was ≥95 %, as estimated by SDS-PAGE and
gel filtration chromatography. To confirm the mutated protein identity, protein bands
corresponding to the mutated LiGLO2 were manually excised from SDS-PAGE gels. Trypsin
digestion and peptide mixture analysis by MALDI-TOF-MS were performed at the EMBL
Proteomics Core Facility.
CChhaapptteerr IIIIII
6644
33..33.. CCrryyssttaalllliizzaattiioonn
Crystallization conditions were obtained for native LiGLO2 with an in house screen of 80
conditions, at 20 °C, applying the hanging-drop vapour-diffusion method using the crystallization
tools from Nextal Biotechnologie. Drops were prepared by mixing 2 µl of protein solution with 2 µl
of each precipitant solution. Several conditions in the crystallization screen produced crystals.
Crystals were obtained in various conditions including PEG ranging from 400 to 8000, MES or
MOPS with pH between 5.5 and 7.0 and sodium or ammonium acetate. The best crystals were
obtained by mixing 1 µl of 25 mg.ml-1 recombinant glyoxalase II in 10 mM Hepes pH 7.0 with 2 µl of
a reservoir solution containing 30 %(w/v) PEG 8000, 0.2 M magnesium chloride and 0.1 M acetate
pH 5.5. This final crystallization conditions resulted in thick plates, which grew within 2 days to
their maximum dimension, approximately 0.16 x 0.06 x 0.02 mm at 15 °C (Figure III.2.).
33..44.. DDaattaa ccoolllleeccttiioonn aanndd ccrryyssttaall ssttrruuccttuurree ddeetteerrmmiinnaattiioonn
All data was collected at 100K. A low-resolution data set was collected in-house, using a
MAR-Research image plate detector. A high resolution native data set was collected at 100K at
beamline ID14-1 and at beamline ID23-1, at the European Synchrotron Radiation Facility (ESRF) in
Grenoble (France) using an ADSC Quantum4R CCD detector. The crystal diffracted beyond 2.15 Å.
The diffraction experiments showed that glyoxalase II crystals belong to space group C2221, with
unit-cell parameters a = 65.7, b = 88.3 and c = 85.2 Å. The data were processed using MOSFLM 6.2.5.
(Leslie 1992) and scaled using SCALA from the CCP4 program package version 6.0 (The CCP4 suite:
programs for protein crystallography 1994). In order to estimate the protein content of the
asymmetric unit, the Matthews coefficient, VM (Matthews 1968) and solvent content were calculated
based on a subunit molecular weight of 32.5 kDa (predicted from sequence). The structures were
solved by molecular replacement using the structure of the human homologue (PDB code 1QH3
(Cameron et al. 1999)) as a search model and the program Phaser (Read 2001) from the CCP4 suite.
Manual model-building was performed using COOT (Emsley & Cowtan 2004). Refmac5
(Murshudov et al. 1998) and all data were used for refinement. TLS refinement cycles (using TLS
Motion Determination (Painter & Merritt 2006) were combined with validation and model-building.
Water molecules were located with COOT and refined using ArpWarp from the CCP4 suite. All
molecular graphic images were generated using the UCSF Chimera (Pettersen et al. 2004).
CChhaapptteerr IIIIII
6655
33..55.. MMeettaall aannaallyyssiiss ooff rreeccoommbbiinnaanntt nnaattiivvee aanndd mmuuttaanntt ggllyyooxxaallaassee IIII
Metal analysis was carried out by Inductively-Coupled Plasma (ICP) emission spectroscopy
(Faires 1993). Both native and mutant protein forms of LiGLO2 were analysed for iron and zinc
using a Jobin Yvon-Ultima ICP spectrometer available at the Atomic Emission Spectrometry Service
(ICP) from the Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal. A total
of 30 crystals of the native protein were harvested, dissolved in 1 ml type I-H2O and analysed. The
mutant LiGLO2 was analysed in solution at a concentration of 50 µg/ml.
33..66.. EEnnzzyymmee aaccttiivviittyy aassssaayyss
High purity methylglyoxal was prepared by acid hydrolysis of methylglyoxal 1,1-dimethyl
acetal, followed by fractional distillation under reduced pressure in nitrogen atmosphere, as
described (Sousa Silva et al. 2005). Oxidised trypanothione (TS2, Bachem) and glutathionyl-
spermidine (GspdSH, Bachem) were reduced with dithiothreitol, in the proportion of 1 mM of thiol
to 3.2 mM dithiothreitol, as reported (Sousa Silva et al. 2005), immediately prior to reaction. S-D-
Lactoyltrypanothione (SDL-TSH) and S-D-lactoylglutathionylspermidine (SDL-GspdSH) were
immediately prepared from the correspondent reduced thiol (T(SH)2 or GspdSH) and
methylglyoxal, using yeast glyoxalase I (2.5 U, Sigma). Methylglyoxal was added in excess (3.34
mM in a 2-ml reaction system), and the hemithioacetal concentration was calculated using the value
of 3.0 mM for the dissociation constant (Vander Jagt et al. 1972). Glyoxalase I assay was performed
at 30 ºC in a 2-ml reaction volume, in 0.1 M potassium phosphate buffer, pH 7.0. The assay was
performed on an Agilent HP 8453 diode array spectrophotometer, with temperature control and
magnetic stirring in the cuvette. The reaction was started by the addition of yeast Glx I. The
formation of the thiolester was followed at 240 nm, and its concentration was calculated using a ε240
of 6.5 mM-1cm-1 for SDL-TSH, a ε240nm of 3.3 mM-1cm-1 for SDL-GspdSH (the same values reported
for bis-lactoylglutathione and for mono-lactoyltrypanothione, respectively (Irsch & Krauth-Siegel
2004)), and a ε240nm of 2.86 mM-1cm-1 for SDL-GSH (Vander Jagt et al. 1975). For the native LiGLO2
concentrations of SDL-T(SH)2 between 0.025 and 0.26 mM, and of SDL-GspdSH between 0.05 and
0.48 mM, were prepared. Activity with SDL-GSH (Sigma) was also assayed for the native form of
the enzyme, with two thiolester concentrations (0.5 and 1.0 mM). For the mutant LiGLO2
concentrations of SDL-T(SH)2 between 0.05 and 0.20 mM, and of SDL-GSH (SDL-GSH, Sigma)
CChhaapptteerr IIIIII
6666
between 0.05 and 1.0 mM were prepared. Glyoxalase I was removed after completing the reaction
using Amicon® Ultra-4 filters (10,000 NMWL, Millipore Corporation). The recovered solution was
used for the activity assay of both forms of L. infantum recombinant glyoxalase II (2 µg), in the same
reaction conditions. The hydrolysis of all thiolesters was followed at 240 nm.
33..77.. DDeetteerrmmiinnaattiioonn ooff kkiinneettiicc ppaarraammeetteerrss
Single-substrate Michaelis-Menten equation parameters for glyoxalase II (Km and V) were
determined by initial-rate and time-course analysis. Non-weighted hyperbolic regression by the
method of least squares was performed with the program Hyper32 (J.S. Easterby, University of
Liverpool, UK, available from: www.liv.ac.uk/~jse/software.html) and the limiting rate was
calculated by time-course analysis (in-house developed software, Ferreira, AEN).
33..88.. AAccttiivviittyy iinn ccrryyssttaallss
Crystals were soaked with the substrate S-D-lactoyltrypanothione. This was prepared from
5 mM of reduced trypanothione and 30 mM of methylglyoxal, in a 0.9-ml reaction system, using
commercial yeast glyoxalase I (10 U). The enzyme was removed using an Amicon® Ultra-4 filter
(10,000 NMWL, Millipore Corporation, Molsheim, France) and substrate solution was concentrated
to 100 mM. Soaking solution contained the substrate (0.5 µl) in 5 µl of 40 % PEG 8000, 2 M MgCl2
and 1M acetate pH 5.5. An intact crystal was placed in each drop of the soaking solution 30 min,
after which they were frozen in liquid nitrogen.
33..99.. EEvvoolluuttiioonnaarryy aannaallyyssiiss
Phylogenetic relationships were inferred from multiple protein sequence alignment by
ClustalW (Thompson et al. 1994), slow/accurate algorithm, using the protein weight matrix Gonnet
series (available at MegAlign from DNASTAR Lasergene package version 7). Protein sequences
used in this analysis were: L. infantum glyoxalase II characterised in this work (GenBank acc. nr.
ABC41261), L. donovani glyoxalase II (AAW52503), L. major putative glyoxalase II (CAJ02466), T.
cruzi glyoxalase II (AAL96759), T. brucei brucei glyoxalase II (CAD37800), H. sapiens glyoxalase II
(CAA62483), A. thaliana glyoxalase II (AAF19564), E. coli probable hydroxyacylglutathione
hydrolase (P0AC84), S. cerevisiae glyoxalase II (CAA71335), Canis familiaris protein similar to
CChhaapptteerr IIIIII
6677
hydroxyacylglutathione hydrolase (XP_537013), Anopheles gambiae putative member of the metallo-
β-lactamase superfamily (XP_310681), Glossina morsitans protein fragment (Gmm-6805), Lutzomyia
longipalpis putative protein containing the conserved domain of β-lactamase superfamily (NSFM-
159f08.p1k), Aspergillus fumigatus hydroxyacylglutathione hydrolase (XP_753369),
Schizosaccharomyces pombe predicted hydroxyacylglutathione hydrolase (NP_588246). G. morsitans
and L. longipalpis protein sequences were deduced from blast searches (protein vs. translated DNA),
using the Blast Server available at the Wellcome Trust Sanger Institute web page (The Pathogen
Sequencing Unit). Sequence alignments and evolutionary analysis was performed by Marta Sousa
Silva, at the Enzymology Group, FCUL, Portugal.
44.. RReessuullttss
44..11.. TThhee LL.. iinnffaannttuumm ggllyyooxxaallaassee IIII ggeennee aanndd ddeedduucceedd pprrootteeiinn
Blast searches in the L. infantum genome on GeneDB (Hertz-Fowler et al. 2004) revealed only
one putative hydroxyacylglutathione hydrolase gene with 888 bp in length, in chromosome 12
(Table III.1). The complete LiGLO2 gene was amplified from genomic DNA of clone
MHOM/MA67ITMAP263 and the sequence was confirmed by sequencing in both directions. The
LiGLO2 sequence (GenBank acc. nr. DQ294972) shows 99.5 % identity to Leishmania donovani
(GenBank acc. nr. AY851655), 96.5 % identity to Leishmania major (GeneDB, LmjF12.0220), and 53.5
% identity with human glyoxalase II (GenBank acc. nr. Q16775).
The deduced protein sequence of 295 amino acid residues clearly identified it as a
hydroxyacylglutathione hydrolase, belonging to the metallo-β-lactamase superfamily. The
predicted molecular mass of 32.5 kDa is comparable to other trypanosomatid enzymes (L. major, L.
donovani and T. brucei), but larger than the 29 kDa human glyoxalase II.
The isoelectric point (pI) of L. infantum glyoxalase II, calculated from the protein sequence, is
6.2 (Table III.1), similar to the 6.0-6.5 from T. brucei (Irsch & Krauth-Siegel 2004), 6.0 for L. donovani
(Padmanabhan et al. 2005) and 6.2 for Arabidopsis thaliana (Crowder et al. 1997). The obtained value
differs from the 8.5 pI for the human glyoxalase II, determined by isoelectric focusing (Ridderstrom
et al. 1996).
CChhaapptteerr IIIIII
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The L. infantum deduced protein contains the highly conserved metal binding motif
THXHXDH, common to all glyoxalases II (Figure III.1.), including human, yeast, Arabidopsis and
also present in the metallo-β-lactamase family, which are known to require Zn(II) (Concha et al.
1996; Crowder et al. 1997; Zang et al. 2001).
Table III.1. Properties of L. infantum glyoxalase II gene and protein.
*Deduced from the DNA or protein sequence. **Experimental data from purified recombinant His-tagged
protein. †Previously obtained value (Sousa Silva et al. 2005).
GenBank accession numbers
Gene: Protein:
DQ294972 ABC41261
Location in the L. infantum genome
Chromosome: 12 Location: bp 98258-99145 Length: 888 bp
mRNA (start and stop codons included) * 888 bp
Amino acids * 295
Isoelectric point (without His-tag) * 6.21
Molecular mass (without His-tag) * 32.5 kDa
Bound metal ion ** Zn2+ and/or Fe2+
Substrate: S-D-lactoyltrypanothione **
Km 0.091 mM
Specific activity 0.497 U.mg-1 enzyme
kcat 1.0x103 s-1
kcat /Km 1.1x107 M-1.s-1
Activity in L. infantum promastigote extracts † 0.18 U.mg-1 protein
Substrate: S-D-lactoyl-glutathionylspermidine **
Km 0.324 mM
Specific activity 6.3 U.mg-1 enzyme
kcat 12.7x103 s-1
kcat /Km 13.9x107 M-1.s-1
CChhaapptteerr IIIIII
6699
L. infantum - - - - M R N Y C T K T F G S A F S V T V V P T - - - - L K D N F S Y L I N D H T T H T L A A V D V 42
L. donovani - - - - M R N Y C T K T F G S A F S P T V V P T - - - - L K D N F S Y L I N D H T T H T L A A V D V 42
L. major - - - - M R N Y C T K T F G S T F S V T V V P T - - - - L K D N F S Y L I N D H T T H T L A A V D V 42
T. cruzi M H T N T M E V V V K H M G A A F S V A V I P V - - - - L K D N Y T Y I I H D K T T N T M A A V D V 46
T. brucei - - - - - M E V V V K S I G T A F T V A V I P V - - - - L K D N Y S Y V I H D K A T N T L A A V D V 41
H. sapiens - - - - - - - - - - - - - - - - M K V E V L P A - - - - L T D N Y M Y L V I D D E T K E A A I V D P 30
C. familiaris - - - - - - - - - - - - - - - - M K V E L L P A - - - - L T D N Y M Y L I I D D E T K E A G V V D P 30
A. thaliana - - - - - - - - - - - - - - - - M K I F H V P C - - - - L Q D N Y S Y L I I D E S T G D A A V V D P 30
A. gambiae - - - - - - - - - - - - - - - - M T V T K I P A - - - - L K D N F M Y L V V C N A T R Q A A V I D P 30
G. morsitans - - - - - - - - - - - - - - - - M Q V K I L P A - - - - L Q D N Y M Y L I I C G S T R E A A I V D P 30
L. longipalpis - - - - - - - - - - - - - - - - M D V K I L P A - - - - L Q D N Y M Y L I V D K A T K D A A I V D P 30
S. cerevisiae - - - - - - - - - - - - - - - - M Q V K S I K M R W E S G G V N Y C Y L L S D S K N K K S W L I D P 34
A. fumigates - - - - - - - - - - - - - - - - M H V Q S I P I - W T G K G N N Y A Y L V T D E P T K E S V I I D P 33
S. pombe - - - - - - - - - - - - - - M S F Q I L P I P M - W V G T Q D N Y A Y L L L C E E T R Q A A I V D P 35
E. coli - - - - - - - - - - - - - - - - M N L N S I P A - - - - F D D N Y I W V L N D E A G R C L I V D P G 30
E. carotovora - - - - - - - - - - - - - - - - M N L I S I P A - - - - L Q D N Y I W L L S N K A N R C V I V D P G 30
L. infantum N A D Y K P I L T Y I E E H L K Q Q G N - A D V T Y T F S T I L S T H K H W D H S G G N A K L K A E 91
L. donovani N A D Y K P I L T Y I E E H L K Q Q G N - A D V T Y T F S T I L S T H K H W D H S G G N A K L K A E 91
L. major N A D Y K P I L T Y I E E H L K Q Q G N - A D V T Y T F S T I L S T H K H W D H S G G N A K L K A E 91
T. cruzi S A D T T P I V D Y V E R I R R S I D E R G A G A M S F S T I F S T H K H W D H A G G N V V L P K A 96
T. brucei S V D I D P V I D Y V R R L G - G V D R - - - - T T D L R T I L S T H K H H D H S G G N I S L Q K K 86
H. sapiens - V Q P Q K V V D A A R K H G - - - - - - - - - - V K L T T V L T T H H H W D H A G G N E K L V K L 69
C. familiaris - V Q P Q K V V E A V K K H G - - - - - - - - - - V R L T T V L T T H H H W D H A G G N E K L V K L 69
A. thaliana - V D P E K V I A S A E K H Q - - - - - - - - - - A K I K F V L T T H H H W D H A G G N E K I K Q L 69
A. gambiae - V E P A R V L E V A R E Q G - - - - - - - - - - C K L N Q L L T T H H H W D H A G G N E A L C E Q 69
G. morsitans - V Y P D T V L Q A V K D E N - - - - - - - - - - V K L K K V L T T H H H W D H A G G N E K L L Q M 69
L. longipalpis - V D P P S V L Q A V E E E K - - - - - - - - - - V N L V K V L T T H H H W D H A G G N E K L V K D 69
S. cerevisiae - A E P P E V L P E L T E D E - - - - - - - - - K I S V E A I V N T H H H Y D H A D G N A D I L K Y 74
A. fumigatus - A N P P E V A P E L D A Q I K A G - - - - - - K I K L S A I V N T H H H W D H A G G N N E M L K H 76
S. pombe - A E V N V V M P I L K K K L K N K - - - - - - E I D L Q A I L T T H H H A D H S G G N L N L K K E 78
E. coli - - D A E P V L N A I A A N N - - - - - - - - - - W Q P E A I F L T H H H H D H V G G - - - V K E L 65
E. carotovora - - E A S P V L N A L D Q N A - - - - - - - - - - L L P E A I L L T H H H N D H V G G - - - V S E I 65
M
2
M
2
M
1
M
1
L. infantum L E A M N S T V P V V V V G G A N D S I P A V T K P V R E G D R V Q V G D - L S V E V I D A P C H T 140
L. donovani L E A M N S T V P V V V V G G A N D S I P A V T K P V R E G D R V Q V G D - L S V E V I D A P C H T 140
L. major L E A M N S M V P V M V V G G A N D G I P A V T K P V R E G D R V Q V G N - L S V E V I D A P C H T 140
T. cruzi L K A A G A F R - - - I I G G V N D N I S G V T Q T V R E G D R L S L G A - L Q V E V L E A P C H T 142
T. brucei L N A M G A F R - - - I I G G A N E P I P G V T E K V R E G D H F S I G E - L K V D V L D A P C H T 132
H. sapiens E S - - - - - - G L K V Y G G D D - R I G A L T H K I T H L S T L Q V G S - L N V K C L A T P C H T 111
C. familiaris E P - - - - - - G L K V C G G D D - R I G A L T Q K V T H L S T L Q V G S - L N V K C L S T P C H T 111
A. thaliana V P - - - - - - D I K V Y G G S L D K V K G C T D A V D N G D K L T L G Q D I N I L A L H T P C H T 113
A. gambiae Y R Q H A D W G Q L T V Y G G D D E R I P G L T N R V G Q D D T F A I G Q - L R V R C L A T P C H T 118
G. morsitans F S - - - - - L P L E V Y G G D N - R I G G L N C M V K Q N D H L K I G N - L D V T C L F T P C H T 112
L. longipalpis F Q K - - - - G P L E V F G G D D - R I G A L T K K V G D G D T F K I G N - L S V R C L F T P C H T 113
S. cerevisiae L K E K N P T S K V E V I G G S K - D C P K V T I I P E N L K K L H L G D - L E I T C I R T P C H T 122
A. fumigatus F G - - - - - - K L P V I G G - R - N C Q S V T Q T P A H G E T F K I G E R I S V K A L H T P C H T 118
S. pombe F P - - - - - - H V T I Y G G - S - D Q N G V S H V L Q D K E T L R I G N - V Q I E A L H T P C H T 119
E. coli V E K F P Q I V - - - V Y G P Q E T Q D K G T T Q V V K D G E T A F V L G - H E F S V I A T P G H T 111
E. carotovora L N H Y P N L P - - - V F G P K E T A K C G A T Y L V E E G N T V S L L N - S E F S V I E V P G H T 111
M
2
L. infantum R G H V L Y K V Q H P Q H P N D G V A L F T G D T M F I A G I G A F F E G D E K D M C R A M E K V Y 190
L. donovani R G H V L Y K V Q H P Q H P N D G V A L F T G D T M F I A G I G A F F E G D E K D M C R A M E K V Y 190
L. major R G H V L Y K V Q H P Q H P N D G V A L F T G D T M F I A G I G A F F E G D E K D M C R A M E K V Y 190
T. cruzi R G H V L Y K V Y H P Q A E K D G V A L F T G D T M F V G G I G A F F E G D A A H M C R A L R K V Y 192
T. brucei S G H V L Y K V Y H P Q K A E N G I A L F T G D T M F V G G I G A F F E G D A V L M C S A L R K V Y 182
H. sapiens S G H I C Y F V S K P G G S E - P P A V F T G D T L F V A G C G K F Y E G T A D E M C K A L L E V L 160
C. familiaris S G H I C Y F V S K P N S S E - P P A V F T G D T L F V A G C G K F Y E G T A D E M Y R A L L E V L 160
A. thaliana K G H I S Y Y V N G K E G E - - N P A V F T G D T L F V A G C G K F F E G T A E Q M Y Q S L C V T L 161
A. gambiae T S H V C Y Y V E G G D R G - - E R A V F T G D T L F L A G C G R F F E G T P D Q M Y D A L I G K L 166
G. morsitans W G H I C Y Y V Q S S T G - - - D R Y V L T G D T L F H G G C G 141
L. longipalpis T G H I C Y Y V Q S N D - - - - E G A V F T G D T L F S A G C G R F F E G T P E Q M Y A A L V D K L 159
S. cerevisiae R D S I C Y Y V K D P T - - T D E R C I F T G D T L F T A G C G R F F E G T G E E M D I A L N N S I 170
A. fumigatus Q D S I C Y Y M Q D - - - - G D E K V V F T G D T L F I A G C G R F F E G N A Q E M H K A L N E T L 164
S. pombe R D S I C F Y A H S - - - - S N E H A V F T G D T L F N A G C G R F F E G T A A E M H I A L N A V L 165
E. coli L G H I C Y F S K P - - - - - - - - Y L F C G D T L F S G G C G R L F E G T A S Q M Y Q S L K K L S 153
E. carotovora S G H I A Y Y N A P - - - - - - - - F L F C G D T L F S A G C G R I F E G T P K Q M Y E S I Q K I A 153
M
1
2
‡
CChhaapptteerr IIIIII
7700
Figure III.1. Sequence comparison between L. infantum and other GLO2 enzymes. The sequences were
aligned using ClustalW (Thompson et al. 1994), available at MegAlign from DNASTAR Lasergene package
version 7. GLO2 sequences used in this alignment are mentioned in section 3.9. from this chapter. Residues in
grey boxes are conserved in at least twelve of the sixteen sequences. Residues of conserved binding motif
THXHXDH are in bold and in a box. Residues responsible for spermidine fixation in L. infantum sequence are
marked (*). M1, M2 and M1,2 indicate the residues in L. infantum binding the metal (Zn or Fe) 1, 2 or both,
respectively. Residues marked with † are responsible for the GSH binding in H. sapiens GLO2. The indicated
secondary structure is LiGLO2.
L. infantum H I H - - - - - - - - K G N D - Y A L D K V T F I F P G H E Y T S G F M T F S E K T F P D R A S D D 231
L. donovani H I H - - - - - - - - K G N D - Y A L D K V T F I F P G H E Y T S G F M T F S E K T F P D R A S D D 231
L. major H I H - - - - - - - - K G N D - Y A L D K V T F I F P G H E Y T A G F M T F S E K T F P D R A S N E 231
T. cruzi N L H - - - - - - - H N A T D K E E A D R R T F V F P G H E Y T V N F L Q F A R D T I P P - A H P D 234
T. brucei N L N G A C E S S A C D A T D V Q K R D N H T Y I F P G H E Y T V N F L R F S R D A L P A - S H P D 231
H. sapiens G R L P - - - - - - - - - - - - - - - - P D T R V Y C G H E Y T I N N L K F A R H V E P G - - - - - 189
C. familiaris G R L P - - - - - - - - - - - - - - - - P D T R V Y C G H E Y T I N T F K F A R H V E P S - - - - - 189
A. thaliana A A L P - - - - - - - - - - - - - - - - K P T Q V Y C G H E Y T V K N L E F A L T V E P N - - - - - 190
A. gambiae S A L P - - - - - - - - - - - - - - - - D D T R V Y C G H E Y A L Q N L R F G H Q V E P D - - - - - 195
G. morsitans 141
L. longipalpis S A L P - - - - - - - - - - - - - - - - D E T K V F C G H E Y T A S N L K Y A K H V E P A - - - - - 188
S. cerevisiae L E T V G - - - - - - - - - - - R Q N W S K T R V Y P G H E Y T S D N V K F V R K I Y P Q - - - - - 204
A. fumigatus A S L P - - - - - - - - - - - - - - - - D D T R V Y P G H E Y T R S N V K F C - - - L - T - - - - - 189
S. pombe S S L P - - - - - - - - - - - - - - - - N N T V I Y P G H E Y T K S N V K F A S - - - - K - - - - - 190
E. coli A L P - - - - - - - - - - - - - - - D D - - T L V C C A H E Y T L S N M K F A L S I L P H - - D - - 182
E. carotovora E L P - - - - - - - - - - - - - - - D D - - T V V C C A H E Y T L S N L R F S N D I W P E - - D - - 182
M
1
*‡
+ ‡
L. infantum L A F I Q A Q R A K Y A A A V K T G - D P S V P S S L A E E K R Q N L F L R V A D P A F V A K M N Q 280
L. donovani L A F I Q A Q R A K Y A A A V K T G - D P S V P S S L A E E K R Q N L F L R V A D P A F V A K M N Q 280
L. major L A F I Q A Q R A K Y A A A V K T G - D P S V P S S L A E E K L Q N L F L R V A D P A F V A K M N Q 280
T. cruzi A T F I A S Q L E R Y K E S V A Q R - K P T V P S T L A E E K R Q N L F L R T C D E S F V R E M K H 283
T. brucei V S F V E A Q L R R Y T E S V A G N - V P T V P S T L A E E K R Q N L F L R T C D E A F V R V M N K 280
H. sapiens N A A I R E K L A W A K E K Y S I G - E P T V P S T L A E E F T Y N P F M R V R E K T V Q Q H A G E 238
C. familiaris N A A V Q E K L A W A K E K Y S I G - E P T V P S T I A E E F T Y N P F M R V R E K T V Q Q H A G E 238
A. thaliana N G K I Q Q K L A W A R Q Q R Q A D - L P T I P S T L E E E L E T N P F M R V D K P E I Q E K L G C 239
A. gambiae N A D T R A L L E R A Q A A D L E G R R A L V P S T I G Q E K R I N V F M R V H Q P A V Q A Y V G K 245
G. morsitans 141
L. longipalpis N P A V E E R I Q W T K E R R D A K - L P T V P S T I G A E K S F N P F M R V N E K S V Q E H A N A 237
S. cerevisiae - V G E N K A L D E L E Q F C S K H E V T A G R F T L K D E V E F N P F M R L E D P K V Q K A A G D 253
A. fumigatus - V S Q S E P I K K L E A Y A N Q H Q Q T Q G K F T I G D E - - - - - - - - - K D P E I Q K K T G K 229
S. pombe - H L Q S E A L N H L E G L C N H N Q F I A G H I T M G Q E K Q F N P F M R V T D P E L Q K H L G L 239
E. coli - L S I N D Y Y R K V K E L R A K N - Q I T L P V I L K N E R Q I N V F L R T E D I D L I N V I N E 230
E. carotovora - P D I E S Y L H K I S Q I R E K S - Q S S L P T T L G L E R R I N L F L R C H E I D L K R K I S N 230
L. infantum G - - - - N A H A L M M Y L Y N A C D 295
L. donovani G - - - - N A H A L M M Y L Y N A C D 295
L. major G - - - - S A H A L M M Y L Y N A C D 295
T. cruzi G D - - - T A E T L M Q H L Y D T C P 299
T. brucei G E - - - T A V K L M D F L Y N T C P 296
H. sapiens T - - - - D P V T T M R A V R R E K D Q F K M P R D 260
C. familiaris T - - - - D P V T T M R A I R K E K D H F K V P R D 260
A. thaliana K - - - - S P I D T M R E V R N K K D Q W R G 258
A. gambiae G - - - - T P L E T M Q A L R A A K D K F 262
G. morsitans 141
L. longipalpis S - - - - D G V T T M G F L R R E K D S F 254
S. cerevisiae T N N S W D R A Q I M D K L R A M K N R M 274
A. fumigatus T - - - - D P V E V M A A L R E M K N A M 246
S. pombe N - - - - D P I K V M D E L R T L K N Q G 256
E. coli E T L L Q Q P E E R F A W L R S K K D R F 251
E. carotovora E P E N I E N W Q V F K M L R S K K D C F 251
‡ *‡
CChhaapptteerr IIIIII
7711
Figure III.2 – Crystals of L. infantum
glyoxalase II.
44..22.. OOvveerr--eexxpprreessssiioonn aanndd ppuurriiffiiccaattiioonn ooff rreeccoommbbiinnaanntt ggllyyooxxaallaassee IIII
The L. infantum LiGLO2 gene was cloned into the pET-28a expression vector and this
construct transformed into E. coli BL21-codon plus cells. Small scale growth cultures were used to
optimise over-expression conditions for His-tagged glyoxalase II. Purification was performed by
metal affinity chromatography and yielded 25 mg of pure protein from 3 litres of culture. The
purity of the recombinant His-LiGLO2 was ≥95 %, as estimated by SDS-PAGE and gel-filtration
chromatography (data not shown), and the apparent molecular weight of 32 kDa was in accordance
with the 32.5 kDa deduced from the protein sequence.
44..33.. FFiinnaall ssttrruuccttuurreess
The purified protein produced thick plate crystals
within 2 days (Figure III.2.) which diffracted beyond 2.15 Å
resolution using synchrotron radiation. The crystals belong to
the space group C2221, with unit-cell parameters a = 65.7, b =
88.3 and c = 85.2 Å. Data-collection and processing statistics
are shown in Table III.2.
After further optimization of the crystal growth process, the L. infantum glyoxalase II structure
with spermidine (LiGLO2-spermidine) was refined against data extending to a dmin of 1.8 Å in space
group C2221, to an R factor of 17.2 % and a corresponding Rfree (Brunger 1992) of 19.6 %. The final
enzyme structure has 287 residues and the Matthews coefficient is 1.95 Å3Da-1, corresponding to a
solvent content of ~37 %, assuming the presence of a single molecule in the asymmetric unit (PDB
entry 2P18). Data-collection and processing statistics are given in table III.3.
0.1 mm
CChhaapptteerr IIIIII
7722
Table III.2. – Crystal data and data-collection statistics for first used crystals. Values in parentheses are for the
highest resolution shell.
Data set In-house ESRF
Space group C2221
Unit-cell parameters (Å) a = 66.6, b = 90.1, c = 85.8 a = 65.7, b = 88.3, c = 85.2
Source in-house CuKα ID14-1
Wavelength (Å) 1.542 0.934
No. of observed reflections 170772 209900
No. of unique reflections 7294 13574
Resolution limits (Å) 30.0 - 2.7 (2.85 – 2.7) 30.0 - 2.15 (2.27 - 2.15)
Redundancy 6.0 (5.7) 5.4 (5.2)
R sym (%) 14.2 (47.7) 10.6 (42.5)
Completeness (%) 99.9 (99.9) 98.4 (98.4)
<I/σ (I)> 5.3 (1.6) 5.7 (1.7)
| |∑ ∑− (j)II(j)(j)I=R iisym / , where Ii(j) is the ith measurement of reflection j and I(j) is the overall
weighted mean of j measurements.
Table III.3. Crystallographic data collection and refinement statistics for further optimised native glyoxalase
II crystals and crystals with the product D-lactate.
Data set LiGLO2-spermidine LiGLO2-D-lactate
Space group C2221
Unit Cell Parameters (Å) a = 66.7, b = 89.0, c = 85.9 a = 67.2, b = 89.1, c = 86.0
Source ID23-1 ID23-1
Wavelength (Å) 0.954 0.954
No. of unique reflexions 23962 20692
Resolution limits (Å) 53.4 – 1.8 53.6 – 1.9
Redundancy 5.7 (5.8) 13.8 (14.1)
Rmerge † (%) 6.9 (46.8) 8.7 (48.8)
Completeness (%) 99.8 (100) 100 (100)
⟨I/σ (I)⟩ 6.5 (1.6) 5.1 (1.5)
† ∑∑∑∑ −ll lerge III=R hhhhhm / , where Il is the lth observation of reflection h and hI is the
weighted average intensity for all observations l of reflection h.
CChhaapptteerr IIIIII
7733
Molecular replacement was done using the human glyoxalase II structure (PDB code 1QH3
Cameron et al. 1999)), which shows 35 % sequence identity with the L. infantum glyoxalase II, as a
search model and the molecular-replacement program Phaser (Read 2001). One clear solution was
obtained and the calculated phases were improved using Pirate (Cowtan 2000). A preliminary Cα
trace was manually built from the search model and is currently being rebuilt using COOT (Emsley
& Cowtan 2004) and refined with Recfmac5 from the CCP4 suite (The CCP4 suite: programs for
protein crystallography).
Associated with each protein molecule there are two metal ions, an acetate molecule and a
spermidine molecule. A total of 188 water molecules were found in the structure. The average
temperature factor of the protein is 23.9 Å2. For the final structure, the rmsd from ideal bond lengths
and bond angles was 0.015 Å and 1.519º respectively, and no outlier residues were found in the
Ramachandran plot.
L. infantum glyoxalase II is a 295 amino acid metalloprotein. Like the previously described
human glyoxalase II structure (Cameron et al. 1999), the L. infantum glyoxalase II monomer is
arranged in two domains. The N-terminal domain, including residues 1-209, has a topology of four-
layered β sandwich with two mixed β sheets of four and seven β-strands flanked by 2 long α-
helices. The first half of the sandwich folds as a ββββαβαββ motif and the second half as a ββββαβ
motif. Thus, the unit ββββαβ is common to the two halves (β1β2β3β4α1β5 and β8β9β10β11α3β12). The C-
terminal domain, including residues 210-295, is an all α-helical domain located at the edge of the N-
terminal domain. Preceding helix α6, a section of chain extends the second sheet of the N-terminal
domain by hydrogen bonding to β11. A β hairpin loop of the N-terminal domain, situated after β11,
protrudes into the C-terminal domain, making hydrogen-bonding and hydrophobic interactions
with residues on helices α4 and α6 (Figure III.3.).
The L. infantum glyoxalase II structure is similar to the whole structure of the human
homologue (PDB 1QH3 (Cameron et al. 1999)), being these structures superimposed with an rmsd
of 1.60 Å (using 191 Cα atoms). Compared to the human glyoxalase II, L. infantum glyoxalase II has
one extra β strand at the N-terminal and an extra α helix at the C-terminal domain. As a
consequence, the referred ββββαβ motif, common to the two halves of the sandwich, is slightly
longer than the human homologue, but identical to the one on other metallo-β-lactamases (Carfi et
al. 1995).
CChhaapptteerr IIIIII
7744
Figure III.3. Comparison of the overall structure and active site of L. infantum and human glyoxalase II. L.
infantum (A, rainbow colour) and human glyoxalase II (grey) superimpose (B) with a root mean square
deviation of 1.60 Å (using 191 Cα atoms); arrows point the β-sheet and α-helix present in LiGLO2-spermidine
and not in the human glyoxalase II. (C) Detail of superposition of the active site of the two structures showing
the high homology of the active site. Metals (M1 and M2) are coordinated by the same residues; identified
residues are from L. infantum. The figure was prepared using UCSF Chimera (Pettersen et al. 2004).
C
A B
M2
M1
Asp80
His139
His78
His76
His81
His210 Asp164
CChhaapptteerr IIIIII
7755
A crystal structure containing D-lactate in the active site (LiGLO2-D-lactate), one of the
reaction products, was obtained after soaking 30 minutes with the substrate (Figure III.4.).
Figure III.4. Detail of the structure of L. infantum with D-lactate (LiGLO2-D-lactate) located at the active site.
Residues from the active site interacting with the metals (M1 and M2) and with the D-lactate molecule are
shown. The figure was prepared in UCSF Chimera (Pettersen et al. 2004).
Hence, the protein remained active in the crystal form. As the previous crystals, this crystal
belonged to the space group C2221, with unit-cell parameters approximately identical to the ones
reported previously: a = 67.2 Å, b = 89.1 Å e c = 86.0 Å. Matthews coefficient was 1.97 Å3Da-1,
corresponding to a solvent content of ~38 %, assuming the presence of a single molecule in the
asymmetric unit (PDB 2P1E). The L. infantum glyoxalase II structure containing D-lactate was
refined against data extending to a dmin of 1.9 Å to an R factor of 18.3 %, and corresponding Rfree of
22.0 %. The final structure containing D-lactate is very similar to the native L. infantum glyoxalase II
model and has an average temperature factor of 26.2 Å2. The average temperature factor of the D-
lactate molecule was 30.1 Å2 and this molecule was located between the two metal ions, separated
by 3.58 Å. It was bound to both metals through its O3 atom, at distances 2.30 Å and 2.68 Å from
metal 1 and 2, respectively. The bridge established by this latter atom replaces the water molecule
present in the LiGLO2-spermidine structure. The D-lactate was also bound to metal 1 through its O1
and O2 atoms, at distances 2.87 Å and 2.14 Å, respectively, which replaces the bridge established by
the acetate molecule oxygen atoms in the LiGLO2-spermidine structure. This is the only major
change provoked by the insertion of D-lactate in the structure. There are a total of 102 water
Asp80
His139
His78
His76
His81
D-Lactate
M1
M2
His210
Asp164
CChhaapptteerr IIIIII
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molecules in this structure. For the final structure, the rmsd from ideal bond lengths and angles
distances was 0.016 Å and 1.540º, respectively, and no outlier residues were found in a stringent
boundary Ramachandran plot.
44..44.. TThhee aaccttiivvee ssiittee
Previous sequence analysis revealed the presence of the highly conserved metal binding
motif THXHXDH in trypanosomatid and all other glyoxalase II proteins (Figure III.1.). The active
site extends across the domain interface and harbours a metal binding-site. There was clear electron
density for a binuclear metal binding-site in the domain interface. The average temperature factor of
each zinc atom was 17 and 18 Å2, whereas for each iron atom was 15 Å2. Metal analysis (ICP)
showed a 1:2 zinc:iron ratio, suggesting that iron could be the main metal present in the active site,
contrary to the results obtained by measuring the temperature factor. Both data indicate that this
enzyme is able to bind either zinc or iron. In T. brucei glyoxalase II, sequence analysis pointed to
zinc as the main metal (Irsch & Krauth-Siegel 2004), as well as metal analysis in glyoxalase II from
A. thaliana, being this zinc:iron binuclear centre, essential for substrate binding and catalysis, was
reported (Zang et al. 2001). Although the human glyoxalase II structure revealed the presence of two
zinc ions, it was also not possible to discriminate between zinc and iron (Cameron et al. 1999).
The two metals from the L. infantum glyoxalase II structure, separated by 3.32 Å, are
coordinated by seven amino acid residues from 3 different regions of the N-terminal domain, and
bridged by an acetate molecule, a water molecule and Asp164. This latter residue seems to be in a
more favourable position for metal 1 coordination than for metal 2, as the Asp164 oxygen atom is
further away from metal 2 (2.51 Å) than from metal 1 (2.09 Å). Metal 1 interacts with Asp80 (2.26 Å),
His81 (2.10 Å), Asp164 (2.09 Å), His210 (2.08 Å), whereas metal 2 is coordinated by His76 (2.27 Å),
His78 (2.13 Å), His139 (2.06 Å) and Asp164 (2.51 Å), (Figure III.5.). This characteristic tetrahedral
coordination to each metal is identical to the one reported to the human glyoxalase II structure
when bound to a glutathione analogue (Cameron et al. 1999).
CChhaapptteerr IIIIII
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Figure III.5. L. infantum glyoxalase II substrate binding site (LiGLO2-spermidine). Detail of the active site
showing the residues interacting with the metals (M1 and M2) and the spermidine molecule (SPD), with
overlaid electron density contoured at 1 σ (omit map computed without both metals and the spermidine
molecule. The figure was prepared in CCP4mg (Potterton et al. 2002; Potterton et al. 2004).
44..55.. TThhee ssuubbssttrraattee--bbiinnddiinngg ssiittee
The thiol replacement in the glyoxalase system of trypanosomatids suggests that these
enzymes are different from all other eukaryotic glyoxalases. Indeed, the human glyoxalase II
structure revealed three conserved basic residues involved in thiolester binding, Arg249, Lys143
and Lys252 (Cameron et al. 1999), also present in almost all eukaryotic glyoxalases II. These residues
are neither found in L. infantum protein, nor in the proteins from T. brucei or other kinetoplastid
organisms (Irsch & Krauth-Siegel 2004). L. infantum glyoxalase II does not use lactoylglutathione as
substrate, as confirmed by direct assay at 240 nm and by the more sensitive DTNB assay. A
spermidine molecule was bound to the recombinant L. infantum enzyme and crystallised close to the
active site. This molecule has an average temperature factor of 25.8 Å2 and interacts with Ile171,
Ala173, Tyr212, Phe219 and Phe266 (Figure III.4.). Curiously, the Phe266 residue, interacting with
CChhaapptteerr IIIIII
7788
the spermidine, is present in the extra α helix at the C-terminal domain from the L. infantum
glyoxalase II. Although it also exists in the human glyoxalase II (Phe224), this residue does not seem
to interact with the substrate in this enzyme.
44..66.. LLiiGGLLOO22 kkiinneettiiccss:: ssppeecciiffiicciittyy ffoorr tthhiioolleesstteerrss ooff SSPPDD--GGSSHH ccoonnjjuuggaatteess
In cell-free extracts, L. infantum glyoxalase II is highly specific for S-D-lactoyltrypanothione
and does not show activity with S-D-lactoylglutathione (Sousa Silva et al. 2005). Structural data
revealed a spermidine molecule accommodated in the substrate-binding site. It might be possible
that the glutathionyl-spermidine derived thiolester is also a substrate. Recombinant LiGLO2 was
assayed for both trypanothione- and glutathionyl-spermidine-derived thiolesters. With
bis(lactoyl)trypanothione as substrate, a Km of 0.091 mM and a kcat of 1.0x103 s-1 were determined
(Table III.1), in good agreement with results obtained in cell free extracts (Sousa Silva et al. 2005).
When the substrate is the glutathionyl-spermidine-derived thiolester, a Km of 0.32 mM and a kcat of
1.27x104 s-1 were obtained (Table III.1). As expected, no activity was detected with S-D-
lactoylglutathione. Results confirmed that the LiGLO2 shows specific affinity towards spermidine-
glutathione conjugate-derived thiolesters. Although kinetics point to the glutathionyl-spermidine-
derived thiolester as the preferred substrate, glutathionyl-spermidine exists in L. infantum in much
lower concentration than trypanothione (unpublished results), being the lactoyltrypanothione the
most probable physiological substrate for LiGLO2.
44..77.. MMuuttaanntt ffoorrmm ooff LLiiGGLLOO22
A mutant form of LiGLO2 was produced in order to evaluate the importance of residues
Tyr291 and Cys294 in the catalytic mechanism of L. infantum glyoxalase II and investigate substrate
specificity in this enzyme. Therefore, these residues were replaced by arginine and lysine,
respectively, by direct mutagenesis.
The purification process of mutated LiGLO2 yielded 11.4 mg of protein per litter of culture,
high enough to make the protein available for further biochemical and structural studies. The
enzyme was purified by cobalt-affinity chromatography and gel filtration, achieving about 95 %
purity and the expected molecular mass, as estimated by SDS–PAGE (Figure III.6.).
CChhaapptteerr IIIIII
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Figure III.6. Analysis of purified recombinant LiGLO2 by SDS-PAGE. From total cell content (T), the soluble fraction
(S) containing expressed protein was loaded into a cobalt-NTA affinity column. Flow-through (FT), eluted fraction (E)
and purified mutated LiGLO2 fractions collected from the gel filtration column (GF Fractions) were analysed by
Coomassie-stained 12 % SDS-PAGE. M, Molecular weight marker (Carl Roth) positions are indicated on the left in kDa.
The ThermoFluor experiment consists on a miniaturised high-throughput protein stability
assay based on monitoring the thermally induced unfolding of a protein in presence of diverse
buffers, by measuring the temperature midpoint of the protein-unfolding transition (Tm)
(Pantoliano et al. 2001). It was a crucial step in mutated LiGLO2 stability improvement, as shown by
protein thermal high instability in different buffers (Figure III.7.). The pH-NaCl concentration
screen performed showed the protein to be most stable in 30 mM Tris pH 8.5 with 100 mM NaCl
(Figure III.7.), while the addictive screen evidenced a significant increase on protein stability when
adding a divalent metal chloride, especially MgCl2, or PEG to the buffer (Figure III.8.). These results
are in agreement with the crystallization condition used for the native LiGLO2, which contained
both MgCl2 and PEG (Trincão et al. 2006).
CChhaapptteerr IIIIII
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Figure III.7. Protein stability accessed by ThermoFluor. Protein stability is higher in Tris-HCl pH 8.5 buffer
with 100 mM NaCl (buffer A) comparing to others assayed. Tm is the temperature midpoint of the protein-
unfolding transition.
Figure III.8. Stability of purified mutant LiGLO2. ThermoFluor screen with different additives showed that
the 50 mM Tris-HCl pH 8.5 buffer containing divalent metal chloride or PEG improved protein stability
relatively to the control buffer without additive (//).
CChhaapptteerr IIIIII
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Protein identification and successfully mutated residues were confirmed by MALDI-FTICR-
MS. A protein band corresponding to the mutated LiGLO2 was excised and digested for peptide
mass fingerprinting analysis. Only 16.0 % of protein sequence was covered, but it included the two
mutated residues (Y291R and C294K), confirming the enzyme’s identity and mutation and
validating the correct expression of the mutant protein.
Pure mutated LiGLO2 showed to be kinetically active when assayed for thiolesters of
trypanothione and glutathione. The respective Km and V parameters are indicated in Table III.4. The
Km was maintained towards S-D-lactoyltrypanothione (0.098 mM) when compared to the native
enzyme (0.091 mM). However, a kcat of 143 s-1 was determined for this substrate, being the specific
activity higher than for the native enzyme (kcat of 16.7 s-1).
In contrast to the native LiGLO2, the mutated form of the protein had activity in presence of S-D-
lactoylglutathione, with a Km of 0.255 mM and a kcat of 695 s-1, showing similar affinity towards this substrate
to the human homologue (Km 0.187 mM, Ridderstrom et al. 1996). This is consistent with the fact that these
two mutated residues are crucial to glutathione binding, being able to shift the enzyme’s specificity and place
its affinity towards SDL-GSH. Mutated LiGLO2 kcat was higher for SDL-GSH than for SDL-T(SH)2, in
agreement with the fact that the enzyme has to catabolise both glutathione moieties of the thiolester derived
from trypanothione.
ICP metal analysis confirmed the presence of zinc and iron, similarly to the native enzyme (Zn:Fe
31:1) (Silva et al. 2008), showing that the mutation did not change metal affinity, an expected result since
mutated residues position far from the metal binding site.
Table III.4. Kinetic parameters for the double mutant and native LiGLO2.
GLO2 Substrate Km
(mM) V
(mmol.s-1.mg-1) kcat (s-1)
kcat /Km (mM-1.s-1)
Mutant LiGLO2 SDL-T(SH)2 0.098 6.93x10-5 143 1.46x10-3
SDL-GSH 0.255 33.6x10-5 695 2.72x10-3
Native LiGLO2a SDL-T(SH)2 0.091 0.828x10-5 16.66 0.18x10-3
SDL-GSH ND ND ND ND Human GLO2b SDL-GSH 0.187 - 780 4.17x10-3
ND – not determined; aValues from Table III.1. from this chapter; bValues from Ridderstrom et al. 1996.
CChhaapptteerr IIIIII
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Glyoxalase II in trypanosomatids evolved to efficiently use a specific and efficient substrate derived
from trypanothione, a unique thiol in these parasites. Residues Tyr291 and Cys294 are conserved in all
trypanosomatid glyoxalase II sequences known to date. Cys294 had been previously identified as one of the
residues responsible for the spermidine binding in LiGLO2 (Silva et al. 2008), suggesting that it has an
important role in the enzyme function. Tyr291 corresponds to Arg249, determinant for glutathione binding in
the human enzyme. Evidence suggests that, the replacement of these two residues in L. infantum by the
human corresponding ones resulted in a change on substrate specificity, showing that these residues are
indeed important for substrate selectivity.
44..88.. TTrryyppaannootthhiioonnee ssppeecciiffiicciittyy:: ssttrruuccttuurraall aanndd eevvoolluuttiioonnaarryy aannaallyyssiiss
Kinetoplastida are protozoan organisms that diverged early in evolution from other
eukaryotes (Hannaert et al. 2003). One particular characteristic shared by theses parasites is the
functional replacement of glutathione by a glutathione-spermidine thiol conjugate, trypanothione
(Muller et al. 2003). Consequently, their enzymes depend on trypanothione and replace the
glutathione-dependent ones from all other eukaryotic organisms and prokaryotes. Examples are the
enzymes of the glyoxalase pathway, which preferably use trypanothione as cofactor, being the
glyoxalase II exclusively dependent on the trypanothione thiolester (Irsch & Krauth-Siegel 2004;
Vickers et al. 2004; Sousa Silva et al. 2005; Ariza et al. 2006).
Trypanosomatid glyoxalases II follow kinetoplastida evolution, diverting early from other
eukaryotic organisms into a separate group (Figure III.9.). The other branch for eukaryotes is
divided into two other major groups, one including fungi and the second containing other
eukaryotes, including trypanosomatid’s hosts (human and dog) and vectors Glossina morsitans
(tsetse fly), Lutzomyia longipalpis (the phlebotomine sandfly responsible for the transmission of
Leishmania in the New World) and Anopheles gambiae (malaria mosquito) proteins (Figure III.9.). The
prokaryotic glyoxalases II analysed, E. coli and Erwinia carotovora, are clearly separated from
eukaryotic proteins, and the highest divergence obtained between them and trypanosomatid
proteins clearly eliminates a prokaryotic origin for the parasite’s enzymes (between 167 and 170 %
divergence, with only 29 % of identity; the percent identity compares sequences directly, without
accounting for phylogenetic relationships, and divergence is calculated by comparing sequence
pairs in relation to the reconstructed phylogeny).
CChhaapptteerr IIIIII
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Figure III.9. Phylogenetic tree of alignment from figure III.1., between L. infantum glyoxalase II and other
glyoxalase II or putative (hydroxyacyl)-glutathione hydrolase enzymes. Rectangular phylogram, performed
from the alignment using MegAlign from DNASTAR Lasergene (branches are proportional to genetic
distance). Species are the same as indicated in Figure III.1.
55.. DDiissccuussssiioonn
On the basis of the three-dimensional structures of the L. infantum (this work) and human
glyoxalases II (Cameron et al. 1999), and taking into account the protein sequence alignment
between other homologous proteins, amino acid substitutions in the various sequences were
analysed in a structural and functional context. In all species, the residues involved in metal binding
are absolutely conserved (Figure III.1., residues marked M1, M2 and M1,2). These residues are
common to all species and correspond exactly to the same metal, 1, 2, or both, in human glyoxalase
II (Cameron et al. 1999). Concerning substrate binding, residue conservation is not observed
between trypanosomatid and other eukaryotic glyoxalase II enzymes. This is an expectable result,
since trypanosomatids use a trypanothione-derived thiolester as substrate, whereas all other
eukaryotic glyoxalases II hydrolyse lactoylglutathione.
Comparing both enzymes (Figure III.10.), differences at the substrate binding-sites are
clearly observed. Trypanosomatids lack the Lys143, Arg249 and Lys252 (Figure III.10b’., in orange)
responsible for the glutathione binding in human glyoxalase II (Cameron et al. 1999). On the other
hand, L. infantum glyoxalase II has the Ile171 (absent from the human enzyme, Figure III.10a’., in
G. morsitans L. longipalpis A. gambiae
H. sapiens
C. familiaris
A. thaliana
S. cerevisiae
S. pombe
A. fumigatus
L. infantum L. donovani L. major
T. cruzi T. brucei
E. coli E. carotovora
Amino Acid Substitutions (x100)
CChhaapptteerr IIIIII
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orange) and both Phe219 and Phe266 strategically positioned to bind the spermidine moiety of the
thiolester (Figure III.10a’.). These two phenylalanine residues, although present in the human GLO2
at positions 182 and 224, respectively, are absent from the substrate binding site. In L. infantum,
superposition of both structures containing the spermidine and the molecule of D-lactate located
between the two metal ions, allow us to speculate on the possible position of the glutathione moiety
of trypanothione (Figure III.10.a., a’.), being clear how the trypanosomatid enzyme evolved to
accommodate the thiolester spermidine moiety.
The crystal structure of glyoxalase II from L. infantum presented here is the first structure
from a trypanosomatid. Previously, only the human (PDB 1QH5), Arabidopsis (PDB 1XM8 and PDB
2Q42) and Salmonella thyphimurium (PDB 2OBW) structures were determined. The most remarkable
feature is the electron density found close to the active centre that is consistent with a spermidine
molecule. This means that the recombinant glyoxalase II was able to bind a molecule that is part of
the physiological substrate. Since the enzyme is absolutely specific for lactoyltrypanthione
substrates and shows no activity with lactoylglutathione, it was possible to elucidate the molecular
mechanism of trypanothione specificity for glyoxalase II. This discovery has far reaching
consequences regarding the design of specific inhibitors for trypanothione dependent enzymes.
Given its uniqueness and importance in trypanosomatids, these enzymes are by far the most
promising therapeutic targets. The sequence and structure of glyoxalase II from L. infantum also
provide hints regarding the evolution of trypanosomatids. They diverged early from other
eukaryotes and are not related at all to prokaryotic enzymes.
The production of a highly pure and active double mutant form of LiGLO2 will allow further
biochemical and X-ray crystallography studies. The future investigation of this mutant enzyme from a
structural point of view will provide a complete understanding of glyoxalase II structure–function
relationship and evolution.
CChhaapptteerr IIIIII
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Figure III.10. Molecular surfaces of L. infantum and human glyoxalase II proteins. (a) Superposition of the
LiGLO2-spermidine (PDB 2P18) with the LiGLO2-D-lactate structure (PDB 2P1E). (a’) Detail of the active and
substrate binding sites, showing the spermidine and D-lactate molecules in the pocket that could easily
harbour the lactoyltrypanothione; metals M1 and M2 are indicated; residues in the pocket are coloured in
yellow and orange; orange coloured residues, Ile171 and Ala173, are absent from the human enzyme. (b)
Human glyoxalase II containing a glutathione molecule (GSH) at the substrate binding site (PDB 1QH5, chain
A). (b’) Detail of the active and substrate binding sites; metals M1 and M2 are indicated; the cavity harbouring
the glutathione (in yellow and orange) is clearly different from the LiGLO2. Orange coloured residues,
Lys143, Arg249 and Lys252, are absent from trypanosomatid enzymes. Figures prepared in UCSF Chimera
(Pettersen et al. 2004).
a. b.
a.’ b.’
GSH M1
M2
D- Lactate
SPD
M1
M2
CChhaapptteerr IIIIII
8866
66.. AAcckknnoowwlleeddggeemmeennttss
The authors thank the European Synchrotron Radiation Facility (ESRF) in Grenoble, France,
for the data collection support. The authors also acknowledge Dr. Maria João Romão for providing
crystallographic data from the Laboratório de Cristalografia de Raios-X at the Faculdade de
Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal.
Work was supported by projects POCTI/ESP/48272/2002 and POCI/QUI/62027/2004 from
Fundação para a Ciência e Tecnologia, Portugal. The authors acknowledge the support from EMBO
short-term fellowship ASTF 318-2008 award and grant SFRH/BD/28691/2006 from Fundação para
a Ciência e Tecnologia, Portugal (both to L. Barata).
Lídia Barata, Marta Sousa Silva, Gonçalo da Costa, António E. N. Ferreira, Linda Schuldt, Ana M. Tomás,
Manfred S. Weiss, Ana Ponces Freire, Carlos Cordeiro. A novel methylglyoxal catabolising aldose reductase
from Leishmania infantum: Co-expression with molecular chaperones and kinetic characterization. Manuscript
in preparation.
CChhaapptteerr IIVV AAllddoossee RReedduuccttaassee ffrroomm LLeeiisshhmmaanniiaa iinnffaannttuumm
CChhaapptteerr IIVV
8899
11.. SSuummmmaarryy
Aldose reductase (EC 1.1.1.21), a NADPH-dependent oxidoreductase, is implicated in the
catabolism of methylglyoxal, a toxic glycolysis by-product. Structure-function analysis of aldose
reductase from Leishmania infantum (LiAKR), a trypanosomatid responsible for visceral
leishmaniasis, by purification of the enzyme following common bacteria systems proved
inappropriate. Soluble and active enzyme could only be obtained upon over-expression in
engineered E. coli strains containing chaperone systems that optimize de novo folding as reported
here.
LiAKR is a newly identified enzyme in this infectious organism. Its cloning, expression,
purification and kinetic characterisation, accomplished for the first time, will be useful to
understand methylglyoxal catabolism in trypanosomatids and to unearth crucial differences
towards its human counterpart.
CChhaapptteerr IIVV
9900
22.. IInnttrroodduuccttiioonn
Leishmaniasis, widespread diseases in the Third World and the Mediterranean basin, affect
nearly 12 million of the world population (Leishmaniasis. Burden of disease, WHO 2010).. Since no
curative drugs or vaccines are available and the existing therapies lead to undesirable side effects
and are limited by the evolution of trypanosomatids’ resistance, effective therapeutic approaches
are much needed. These may rely on unique biochemical characteristics which set trypanosomatids
apart from all other eukaryotic cells. One of the most specific biochemical feature is the functional
replacement of glutathione by trypanothione (N1,N8-bis(glutathionyl)spermidine) (Flohé et al. 1999;
Muller et al. 2003). One of these trypanothione-dependent systems is the glyoxalase pathway,
deemed essential for methylglyoxal catabolism. Since methylglyoxal is toxic and an unavoidable
non-enzymatic by-product of glycolysis (Leoncini et al. 1980; Westwood et al. 1997), hampering the
glyoxalase pathway might cause a methylglyoxal concentration increase and the selective death of
the parasites.
However, the glyoxalase pathway is not the only catabolic route for methylglyoxal in
eukaryotic cells. Aldose reductase (EC 1.1.1.21) was found to be equally important for
methylglyoxal detoxification in other organisms, namely human and yeast (Gomes et al. 2005). This
enzyme reduces methylglyoxal to 1,2-propanediol in a NADPH-dependent two-steps reaction
(Keegan & Blum 1995). A sequence homology based search for the human and yeast aldose
reductase homologue in the L. infantum genome revealed a suitable gene candidate, named L.
infantum aldo-keto reductase (LiAKR). In this work, the identified LiAKR gene was cloned into a
suitable expression vector for recombinant protein expression in E. coli. Attempts to obtain pure
recombinant LiAKR were hampered due to the formation of insoluble inclusion bodies when
expressing the protein in different E. coli strains. This problem was overcome by co-expressing
LiAKR in engineered E. coli strains that simultaneously overproduce different chaperone
combinations (de Marco et al. 2007). The LiAKR was produced in a soluble, enzymatically functional
and stable protein, enabling the realization of further biochemical and future structural studies.
CChhaapptteerr IIVV
9911
33.. MMaatteerriiaallss aanndd MMeetthhooddss
33..11.. CClloonniinngg ooff LLiiAAKKRR
The LiAKR gene was amplified by PCR (Polymerase Chain Reaction) from L. infantum
genomic DNA using as specific forward and reverse primers: 5’
CCGCGCACATATGTCCGCCAACGTGCT 3’ and 5’CACCGCTCGAGTCAAGAACGCGGCGATG
3’, containing NdeI and XhoI site (underlined), respectively. PCR was performed using PWO
polymerase (Promega) under standard conditions (94 °C for 2 min, 55 °C for 1 min, 72 °C for 1 min
(2 cycles); 94 °C for 45 sec, 65°C for 1 min, 72 °C for 1min (30 cycles); 72 °C for 10 min; 4°C). The
purified PCR product was digested by NdeI and XhoI and ligated to the similarly restricted pET-
28a expression vector (Novagen), giving rise to pET28a/His6-LiAKR. Final protein sequence was
preceded by MGSSHHHHHHSSGLVPRGSH, containing a His6-tag (bold) and a Thrombin cleavage
site (underlined). Plasmid purification was performed using standard protocols.
33..22.. EExxpprreessssiioonn ooff LLiiAAKKRR iinn ddiiffffeerreenntt EE.. ccoollii ssttrraaiinnss aanndd pprrootteeiinn ssoolluubbiilliittyy
E. coli strains BL21-codon Plus, BL21 (DE3), BL21 (DE3)-codon Plus-RIL, BL21 (DE3)-codon
Plus-RP, BL21 (DE3) pLysS, BL21 (DE3) star, Rosetta (DE3), Rosetta (DE3) pLysS and five E.coli
BL21 (DE3) strains containing a different chaperone system (DnaK/DnaJ/GrpE,
DnaK/DnaJ/GrpE/ClpB, GroESL, low and high expression DnaK/DnaJ/GrpE/ClpB/GroESL (De
Marco et al. 2007)) were used to analyse the over-expression potential of LiAKR. Trial liquid
cultures of these strains harbouring pET28a/His6-LiAKR were grown at 37 °C in LB (Luria-Bertani)
medium, supplemented with 30 µg/ml kanamycin (Carl Roth) and the adequate antibiotics for
each strain (34 µg/ml chloramphenicol (Carl Roth) and/or 30 µg/ml spectinomycin (Sigma-
Aldrich)) until an OD600nm of 0.6 was reached. LiAKR expression was induced by addition of IPTG
(isopropyl-β-D-thiogalactopyranoside, Carl Roth). Expression temperature and growth time were
optimised (15 °C and 20 °C overnight, 28 °C for 1.5 h and 3.0 h, and 37 °C from 1 to 4 h), as well as
the induction levels by varying IPTG concentration (0.2, 0.5 and 1.0 mM). Cells were harvested,
suspended in buffer A (50 mM Tris pH 7.4, 200 mM NaCl, 2 mM β-mercaptoetanol, all from Carl
Roth) and lysed by sonication. Soluble and insoluble fractions of cell lysates were recovered by
CChhaapptteerr IIVV
9922
centrifugation at 3000 xg, 4 °C, 10 min and analysed for LiAKR expression and solubility using a 12
% SDS-PAGE.
33..33.. PPuurriiffiiccaattiioonn bbuuffffeerr ooppttiimmiizzaattiioonn bbyy TThheerrmmooFFlluuoorr
Purification buffer A was optimised in a ThermoFluor experiment (Ericsson et al. 2006). 5 µl
of 50 × Sypro Orange (fluorophore, Invitrogen), 5 µl of 1 mg/ml LiAKR and 40 µl of each buffer
were added to the wells of a 96-well thin-wall PCR plate (Bio-Rad). Fluorescence was detected
while heating the plate from 4 to 95 °C in increments of 0.5 °C using a ThermoFluor ICycler IQ
thermal cycler (Bio-Rad). Two screens were performed: a pH versus NaCl concentration screen and
an additives screen, including various compounds (nucleotides, metal ions, salts and others)
supplemented to the basis buffer 50 mM Tris-HCl pH 8.5.
33..44.. LLaarrggee ssccaallee pprroodduuccttiioonn aanndd ppuurriiffiiccaattiioonn ffrroomm ssoolluubbllee ffrraaccttiioonn
For protein over-expression, E. coli BL21 (DE3) expressing the chaperone system GroESL and
transformed with pET28a/His6-LiAKR was cultured in 750 mL of LB medium containing
kanamycin (30 µg/mL), chloramphenicol (34 µg/mL) and spectinomycin (30 µg/mL) at 37 °C, until
the culture reached an OD600nm of 0.6. Protein expression was induced with 0.2 mM IPTG at 20 °C,
overnight. Cells were harvested by centrifugation at 7800 xg for 30 min at 4 °C, suspended in 10 ml
of buffer B (50 mM Tris pH 8.5, 200 mM NaCl, 2 mM β-mercaptoethanol, 10 % (v/v) glycerol) per g
of wet cell pellet and lysed by sonication. The soluble fraction obtained by centrifugation at 38000
xg for 40 min at 4 °C was filtered through a 0.22 µm Millex membrane filter (Millipore), before being
applied to a Co-NTA beats (Qiagen) column for purification at room temperature. LiAKR was
eluted in buffer B supplemented with 500 mM imidazole. Fractions containing LiAKR (as confirmed
by 12 % SDS-PAGE analysis) were applied to a gel filtration column (Superdex 75, Amersham
Biosciences) and eluted with buffer B. Vivaspin15 (molecular weight cutoff 30000) concentrator
(Sartorius Stedim Biotech) was used to concentrate the protein. Protein concentration was
determined using a Peqlab Biotechnologie GmbH NanoDrop® ND-1000 Spectometer at 280 nm,
considering an aldose reductase extension coefficient of 42400 M-1 cm-1, as calculated by the
ProtParam tool (Gasteiger E., et al., The Proteomics Protocols Handbook, Humana Press (2005)).
CChhaapptteerr IIVV
9933
33..55.. PPrrootteeiinn aannaallyyssiiss bbyy mmaassss ssppeeccttrroommeettrryy
Protein bands corresponding to the putative LiAKR were manually excised from gels and
digested with trypsin (modified porcine trypsin, sequencing grade, Promega), as previously
reported (Shevchenko et al. 2006). Peptide mixtures were analysed by MALDI-FTICR-MS in a
Bruker Apex Qe 7 Tesla magnet Monoisotopic peptide masses were determined using the Snap 2.0
algorithm in Data analysis 4.0 software (Bruker Daltonics). The Mascot search engine
(www.matrixscience.com) was used for protein identification. This mass spectrometry analysis was
performed by Gonçalo da Costa at the FTICR and Advanced Proteomics Laboratory, FCUL,
Portugal.
33..66.. RReeccoommbbiinnaanntt LLiiAAKKRR aaccttiivviittyy aassssaayy
Recombinant LiAKR activity was assayed by following NADPH oxidation at 340 nm in the
presence of methylglyoxal (Gomes et al. 2005). Assays were performed by varying NADPH
concentration between 0.001 and 0.025 mM at a constant methylglyoxal concentration (5 mM), and
varying methylglyoxal concentration (0.2 to 5 mM) at a constant NADPH concentration (0.025 mM).
Additionally, an assay using methylglyoxal and NADH was performed, and the known substrate
glyceraldehyde was also used in the presence of NADPH. All reactions were monitored at 30 °C in
1.5 mL of 25 mM potassium phosphate buffer pH 7.4, on an Agilent HP 8453 diode array
spectrophotometer, with stirring. NADPH concentration was calculated using the ε340 nm of 6.22 mM-
1.cm-1 and its auto-oxidation rate was considered in all reactions monitored. Km values for NADPH
and methylglyoxal and the limiting rate were calculated by time-course analysis (Ferreira, A.E.N.,
in house developed software). Enzyme stability was also investigated by monitoring the activity of
aliquots of LiAKR stored at 4 ºC, -20 ºC and -80 ºC. High-purity methylglyoxal was prepared by acid
hydrolysis of methylglyoxal 1,1-dimethyl acetal (Sigma-Aldrich), as described previously (Sousa
Silva et al. 2005).
33..77.. LLiiAAKKRR ccrryyssttaalllliizzaattiioonn ttrriiaallss
Commercially available crystal screens and the sitting-drop vapor-diffusion method were
used to perform initial crystallization screening, at the High Throughput Crystallization facility at
CChhaapptteerr IIVV
9944
the EMBL-Hamburg Outstation (Mueller-Dieckmann, 2006). Drops contained 0.5 µl of protein
solution at 20 mg/ml and 0.5 µl of reservoir solution and were equilibrated over 80 µl reservoir
solutions in 96-well format Greiner plates. Crystal needles were obtained (Figure IV.1a.) from the
crystallization condition 1.5 M sodium malonate pH 6. The hanging-drop vapour-diffusion method
was applied using the tools from Nextal Biotechnology to further improve the crystals. Drops were
prepared by mixing 2 µl protein solution (20 mg/ml) with 2 µl reservoir solution containing 1.2 M
sodium malonate pH 5.5, and equilibrated over 1 ml reservoir solution.
44.. RReessuullttss aanndd ddiissccuussssiioonn
44..11.. CClloonniinngg,, eexxpprreessssiioonn aanndd ppuurriiffiiccaattiioonn ooff LLiiAAKKRR
A BLAST search was performed on the L. infantum genome database (GenBank accession no.
AM502219:AM502254) and a gene coding for an aldo-keto reductase-like protein, present in L.
infantum chromosome 27 , was identified and isolated from L. infantum genomic DNA. The LiAKR
gene (GenBank accession no. FJ489648) shows 51 % sequence identity with human aldose reductase
mRNA (AKR1B1, GenBank accession no. NM001628) and 43 % sequence identity with Gre3 from S.
cerevisiae (Locus tag YHR104W), both coding for aldose reductases involved in methylglyoxal
catabolism. The deduced protein, with 372 residues and an apparent molecular weight of 41034 Da,
shares 37 % and 29 % sequence identity with human aldose reductase and yeast Gre3, respectively.
Standard expression techniques, using pET28a expression system in different E. coli strains and a
wide range of induction conditions (IPTG concentration, temperature and induction duration), were
not successful (Figure IV.1.).
Figure IV.1. LiAKR expression conditions trial.
Coomassie-stained 12 % SDS-PAGE shows total crude
lysates of cultures grown at 37 °C for 3h carrying pET28a-
LiAKR (T), fractionated as insoluble (I) and soluble (S)
under culture conditions containing 0.2 and 0.5 mM
IPTG. A culture sample was collected before induction
(T0) for control. Molecular mass marker (M) positions are
given in kDa.
CChhaapptteerr IIVV
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LiAKR appeared only in the insoluble fraction. Protein solubilisation required the use of 8 M
urea and produced an unstable enzyme (data not shown), unsuitable for biochemical studies. The
protein was expressed in a ∆GRE3 yeast mutant, lacking methylglyoxal dependent aldose
reductase activity, being possible to obtain an active enzyme in yeast cell free extract (data not
shown). This expression system revealed the likely need of a more complex protein producing and
processing machinery, including chaperones. This led us to follow a strategy involving LiAKR over-
expression in E. coli strains engineered to co-ordinately overproduce different chaperone systems
(DnaK/DnaJ/GrpE, DnaK/DnaJ/GrpE/ClpB, GroESL, and low and high expression
DnaK/DnaJ/GrpE/ClpB/GroESL (De Marco et al. 2007)). Significant quantities of soluble active
enzyme were produced when using any of the chaperone systems (Figure IV.2.).
Figure IV.2. Chaperone co-expression effect on LiAKR over-expression (GroESL (1), low expression
DnaK/DnaJ/GrpE/ClpB/GroESL (2), DnaK/DnaJ/GrpE (3), DnaK/DnaJ/GrpE/ClpB (4) and high
expression DnaK/DnaJ/GrpE/ClpB/GroESL (5)). Crude lysates of cultures grown at 20 °C overnight
carrying pET28a-LiAKR (T), fractionated as insoluble (I) and soluble (S) under culture conditions containing
0.5 mM IPTG. A culture sample was collected before induction (T0) for control. Molecular mass marker (M)
positions are given in kDa and the arrow indicates LiAKR expression.
E. coli co-expressing the GroESL chaperone system was used for large-scale improved
expression and LiAKR protein was purified to near homogeneity as shown by SDS-PAGE analysis
(Figure IV.3.). The yield was about 30 mg of protein per litter of cell culture, allowing us to proceed
to biochemical studies.
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9966
Figure IV.3. Analysis of purified recombinant LiAKR.
From total cell content (T), the soluble fraction
containing expressed protein (S) was loaded into a Co-
NTA beats column. Both flow-through (FT) and eluted
fraction (E) were analysed by Coomassie-stained 12 %
SDS-PAGE, as well as the highly purified LiAKR
fractions collected from the gel filtration
chromatography (GF Fractions).
44..22.. PPuurriiffiiccaattiioonn bbuuffffeerr ooppttiimmiizzaattiioonn bbyy TThheerrmmooFFlluuoorr
The purification buffer was optimised by ThermoFluor (Ericsson et al. 2006), a miniaturised
high-throughput protein stability assay based on monitoring the thermally induced unfolding of a
protein in presence of diverse buffers by determining the temperature midpoint of the protein-
unfolding transition curve (Tm) (Pantoliano et al. 2001). ThermoFluor investigation combined with
observation of protein behaviour in the buffer during the purification process provided a detailed
picture of the linkage between buffer, additives and protein stability. The systematic study of buffer
effects on protein stability is a very useful tool in purification process optimization.
A pH-NaCl concentration screen was performed showing that the protein was most stable in
50mM Tris pH 8.5 with 200 mM NaCl (Figure IV.4.). The additive screen showed a significant
increase on protein stability when glycerol was added to the buffer (Figure IV.5.). Based on these
results, buffer B (50 mM Tris pH 8.5, 200 mM NaCl, 2 mM β-mercaptoethanol, 10 % (v/v) glycerol)
was selected for the purification procedure.
Figure IV.4. Protein stability accessed by
ThermoFluor. Tris-HCl pH 8.5 with 200
mM NaCl (buffer B) is the buffer allowing
higher protein stability. Tm is the
midpoint of temperature of the protein-
unfolding transition.
CChhaapptteerr IIVV
9977
Figure IV.5. LiAKR stability. ThermoFluor screen with
different additives showed that glycerol improves LiAKR
stability relatively to the control buffer without additive (//).
44..33.. PPrrootteeiinn aannaallyyssiiss bbyy mmaassss ssppeeccttrroommeettrryy
A protein band corresponding to the purified recombinant protein was excised and digested
for peptide mass fingerprinting analysis. A significant MASCOT score was obtained, with 40 % of
LiAKR sequence coverage, thus confirming the enzyme identity and validating the correct
expression of the protein.
44..44.. AAccttiivviittyy ooff rreeccoommbbiinnaanntt LLiiAAKKRR
Recombinant LiAKR was assayed for activity. Following the NADPH oxidation, in presence
of methylglyoxal, a Km for methylglyoxal of 0.59 mM and a Km for NADPH of 0.0022 mM were
determined. kcat/Km values of 0.21 mM-1s-1 and 55.6 mM-1s-1 for methylglyoxal and NADPH,
respectively, were obtained. Additionally, no activity was observed when using methylglyoxal and
NADH. DL-glyceraldehyde was also a substrate for this enzyme, using NADPH as reported for
other aldose reductase enzymes (Ryle & Tipton 1985; Grimshaw et al. 1990; Del Corso et al. 1990).
The effect of storage on enzyme activity was investigated again for the following conditions:
at 4 °C, -20 °C and -80 °C, for 1 day, 1 week and 1 month. LiAKR showed to be highly stable and
functionally active when conserved at -20 °C or -80 °C (data not shown).
CChhaapptteerr IIVV
9988
44..55.. PPrreelliimmiinnaarryy ccrryyssttaalllliizzaattiioonn ttrriiaallss
Our purpose is to investigate LiAKR from a structural point of view, to obtain a complete
understanding of this important methylglyoxal catabolising enzyme and to evaluate its potential as
an anti-leishmaniasis drug target.
The first crystallization screens for the purified recombinant LiAKR protein were successful.
Crystals needles were grown (Figure IV.6.a.). Crystal optimization resulted in rod-shape crystals
(Figure IV.6.b.) which grew within 2 days at 20°C. LiAKR crystals did not diffract at any of the
synchrotrons ESRF-Grenoble or DESY-Hamburg. Further crystal optimization is still required.
Figure IV.6. LiAKR crystals. (a) Crystal
needles from the initial screen. Picture
from the High Throughput
Crystallization facility robot, EMBL-
Hamburg. (b) Improved crystals by
vapour-diffusion method.
55.. CCoonncclluuddiinngg rreemmaarrkkss
In the present work, highly pure, active and stable trypanosomatid aldose reductase was
purified for the first time. The production yield in E. coli co-expressing chaperones is large enough
to allow further X-ray crystallography and biochemical analysis. This novel L. infantum enzyme
was kinetically characterised with methylglyoxal and NADPH as substrates. We are currently
investigating LiAKR from a structural point of view, already having preliminary crystallization
results. We look forward to obtain a detailed understanding of this important methylglyoxal
catabolising enzyme and to evaluate its potential as an anti-leishmaniasis drug target.
CChhaapptteerr IIVV
9999
66.. AAcckknnoowwlleeddggmmeennttss
The work was supported by projects POCTI/ESP/48272/2002, POCI/QUI/62027/2004 and
REDE/1501/REM/2005 from Fundação para a Ciência e Tecnologia, Portugal. The authors
acknowledge the support from EMBO short-term fellowship ASTF 318-2008 award (to L. Barata)
and grants SFRH/BD/28691/2006 (to L. Barata) and PDTC/QUI/70610/2006 (to G. Costa) from
Fundação para a Ciência e Tecnologia. We thank Dr. Arie Geerlof (EMBL-Hamburg) for providing
the E. coli BL21 Star (DE3) chaperone system cells.
CChhaapptteerr VV CCoonncclluuddiinngg RReemmaarrkkss
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CCoonncclluuddiinngg RReemmaarrkkss
Leishmaniasis have long been considered neglected diseases for the poor investment in
research on the development of both vaccines and drugs, although it is directly responsible for the
death of 57000 people per year (WHO 2008). Presently there are no vaccines against these diseases,
and both visceral and cutaneous leishmaniasis are traditionally treated with pentavalent
antimonials, which are toxic especially in the presence of HIV co-infections. The latter co-infections
add an increased risk associated to leishmaniasis both in developed countries and in the third
world. Additionally, resistance to these compounds has been reported. For instance, antimonial
resistance has strongly arisen in India, where visceral leishmaniasis is endemic (Sundar 2001). There
are some alternative drugs: Amphotericin B, requiring intravenous administration; Pentamine, less
and less effective; and Miltefosine, the only oral antileishmanial agent. However, these drugs’
inefficiencies, high costs and emergence of resistant trypanosomatids demand the development of
new therapeutic approaches towards these parasites. Considering that the most adequate
therapeutic targets are likely to be based on biochemical differences between parasite and host,
trypanothione metabolism and dependent enzymes, which exist only in Kinetoplastida, arise as
promising candidates.
Invasion and persistence of L. infantum within macrophages is ensured by several survival
strategies. One of these is the anti-oxidant defence system involving the unique thiol trypanothione.
Another important trypanothione function is the detoxification of ketoaldehydes such as
methylglyoxal (Krauth-Siegel et al. 2005), an unavoidable toxic compound for all living cells. The
most common methylglyoxal formation pathway is the often overlooked chemical instability of the
glycolytic dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate (Lohman & Meyerhof
1934). Concerning methylglyoxal detoxification, multiple catabolic pathways have been described,
being the glyoxalase system and the aldose reductase the most important and physiologically
relevant. The glyoxalase pathway catalyses the formation of D-lactate from methylglyoxal, being
considered the main pathway involved in this compound’s detoxification. The aldose reductase,
another methylglyoxal catabolising enzyme, gains importance in some organisms’ life stages
(Samaras et al. 1988).
The glyoxalase pathway was studied in L. infantum protein extracts and its potential as a
drug target was evaluated by modelling and computer simulation (Sousa Silva et al. 2005). Despite
the findings revealing that this pathway might be a poor drug target, this system is still considered
CChhaapptteerr VV
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a potential antileishmanial therapeutic target by some authors (Fairlamb et al. 2006; Padmanabhan
et al. 2005; Padmanabhan et al. 2006; Opperdoes & Michels 2008). In these studies, however,
methylglyoxal catabolism through the enzyme aldose reductase or the methylglyoxal flux
distribution between both pathways was not considered. Hence, the main objective of the present
work was to study both systems from L. infantum. The structure determination of all three enzymes
involved in methylglyoxal catabolism, together with their detailed kinetic characterization and
complementary biochemical studies, allowed a comprehensive recognition of the enzymes in L.
infantum and likely place us a step further in this parasite knowledge.
In the present work, glyoxalase I, glyoxalase II and aldose reductase from L. infantum were
kinetically and structurally characterised. The three proteins were over-expressed, purified and
further studied in detail by X-ray crystallography, enzyme kinetics and biochemical analysis.
Available kinetic data and X-ray structures from other organisms’ homologues were used for
comparison and also as structural search models. Relevant glyoxalase I X-ray structures used were
from L. major (LmGLO1; PDB entry 2C21 (Ariza et al. 2006)), H. sapiens (HsGLO1; PDB entries 1FRO;
(Cameron et al. 1997, 1999; Ridderstrom et al. 1998)) and E. coli (EcGLO1; PDB entries 1F9Z; (He et al.
2000)). Concerning the glyoxalase II, only the human structural model (HsGLO2; PDB entry 1QH5;
(Cameron et al. 1999)) was used in structure analysis. Several aldose reductase human structures
have been determined considering its high impact in diseases like diabetes (HsAKR; PDB entries
1ADS (Wilson et al. 1992); 1ABN (Borhani et al. 1992)), useful in future structure determination and
comparison.
L. infantum GLO1 was identified and both kinetic and structural studies were undertaken.
This is the first detailed report of GLO1 in this parasite, and this study allowed a throughout
comparison to the human and the L. major homologues. The L. infantum and L. major (Ariza et al.
2006) GLO1 structures revealed to be very alike, as expected for the high sequence homology
observed (97 % identity). Furthermore, LiGLO1 showed to preferentially use trypanothione,
similarly to LmGLO1. Despite these similarities, LiGLO1 showed a different metal-content,
relatively to its L. major counterpart. Based on the metal-content of the available GLO1 structures, it
was assumed (Vickers et al. 2004) that prokaryotic enzymes are Ni2+-dependent, while eukaryotic
GLO1 contain Zn2+ at the active site. Eukaryotic Zn2+-dependent enzymes include H. sapiens
(Ridderstrom et al. 1998) and S. cerevisiae (Aronsson et al. 1978). Prokaryotic Ni2+-dependent GLO1
include E. coli, Pseudomonas aeruginosa, Yersinia pestis and Neisseria meningitides (Sukdeo et al. 2004)
enzymes. However, this topic is highly controversial since some enzymes do not follow this pattern,
CChhaapptteerr VV
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as Pseudomonas putida (prokaryotic) GLO1 was reported to be Zn2+-dependent (Saint-Jean et al.
1998), and LmGLO1 (eukaryotic) shown to contain divalent nickel at the active site (Ariza et al.
2006). In spite of its similarities to LmGLO1, LiGLO1 has zinc at its active site, like other eukaryotic
GLO1 enzymes. Metal content in LiGLO1 was determined by ICP analysis, fluorescence-based
metal analysis and anomalous scattering. These methodologies allowed the metal identification and
quantification. LiGLO1 zinc-dependence would suggest a later divergence from the other
trypanosomatid throughout evolution, consistent with sequence data analysis. However, a detailed
metal analysis of GLO1 from other trypanosomatids would be required to validate this hypothesis.
L. major and L. infantum are far enough in evolution to cause different forms of Leishmaniasis, being
understandable to find differences in their respective biochemistry, resulting from different
evolutionary pressures. The importance of studying LiGLO1 was undoubtly enhanced by this
enzyme uniqueness revealed in the present work.
Glyoxalase II from L. infantum is absolutely specific towards the thiolester derived from
trypanothione. Its native structure was the first GLO2 structure reported from a trypanosomatid.
When compared to the human enzyme, their general structures are very similar, being the metals at
the active site coordinated by the same residues. The native LiGLO2 structure superimposed to a
LiGLO2 D-lactate containing structure at the active site, obtained by a crystal-soaking with
substrate, showed a distinctive substrate binding pocket relatively to its human homologue, a very
attractive feature for further docking studies. A spermidine molecule was serendipitously co-
purified with the native LiGLO2, allowing the identification of the substrate interacting residues. It
is noteworthy that the LiGLO2 residues involved in S-D-lactoyltrypanothione binding, through its
spermidine moiety, are absent from the human homologue. Furthermore, the human enzyme
residues binding the S-D-lactoylglutathione are absent from the LiGLO2 protein. These results
steered to the step of mutating two of the LiGLO2 residues involved in the substrate-binding pocket
(Tyr291 and Cys294), by the correspondent human enzyme residues, interacting with the
glutathione moiety (Arg249 and Lys252, respectively). The mutation caused protein structural
stability loss, a hardship in achieving the mutated LiGLO2 purification and further crystallization.
Pure recombinant mutated LiGLO2 was finally obtained after optimization of many purification
conditions, especially buffer optimization through ThermoFluor experiments. The kinetic analysis
of the mutated enzyme, performed immediately after purification to avoid protein precipitation,
revealed a substrate shift for this enzyme. The mutation of only two residues of the substrate
binding pocket was not enough to completely shift the enzyme’s specificity towards the
trypanothione-derived thiolester. However, a loss of affinity was observed for the Leishmania
CChhaapptteerr VV
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glyoxalase II physiological substrate, with an increased affinity for the S-D-lactoylglutathione,
showing that the chosen residues are crucial for substrate selectivity.
In this work, the first Leishmania aldose reductase (LiAKR) was identified in the genome and
the recombinant enzyme was made available for further biochemical and structural studies.
Previously, a gene from L. major coding for a protein from the reductase superfamily was reported
(Samaras et al. 1988). Also, activity of a methylglyoxal reductase and an aldehyde reductase from L.
donovani was detected (Goshal et al. 1989; Keegan & Blum 1995). Recently, it was described the
identification and purification of a L. donovani prostaglandin-f2-alpha-synthase (Rath et al. 2009),
another enzyme included in the AKR superfamily, incorrectly classified as an aldose reductase. The
LiAKR gene codes for an aldose reductase involved in methylglyoxal catabolism. Its hampered
expression was overcame by co-expression with different chaperone systems in E. coli. Additionally,
ThermoFluor experiments were critical for buffer optimization and purification of pure and active
protein in large amounts. The recombinant enzyme was characterised by kinetic analysis and
identified by mass spectrometry. LiAKR crystals were also grown, although further optimization of
this process is required for structure determination.
Indeed, protein structure determination by X-ray crystallography is a long process and not
fully automatic. The protein purity is critical to protein crystal’s growth and its conditions
optimization is not always trivial. In the present project, these methods took much longer that
foreseen for the mutated LiGLO2 and the aldose reductase, postponing these structures’ solution.
From a future perspective, it would be interesting to obtain the mutated LiGLO2 structure model, in
order to observe the structural basis for this enzyme’s affinity change towards the glutathione-
derived substrate. Aldose reductase crystals still require further optimization for a better diffraction
and structure solution. Despite LiAKR catabolising the same substrates as the human aldose
reductase, their sequence identity of only 51 % attaches an additional interest to solving the
parasite’s enzyme structure and compare both proteins. Moreover, it would be interesting to
perform drug-design studies in all three enzymes’ structures. The different potential inhibitors
would be used in kinetic analysis and further co-crystallization experiments.
L. infantum methylglyoxal catabolising systems seem to be quite robust and controlled.
Studies in the group revealed that enzymes’activities vary along the parasite’s life cycle. In total
protein extracts of promastigotes (either exponential or stationary phase) and amastigote forms of L.
infantum, specific activity of both glyoxalase enzymes increase during stationary growth phase and
further in amastigote forms of the parasite (M. Sousa Silva, data not published). Aldose reductase
CChhaapptteerr VV
110077
shows an opposite behaviour, as its specific activity is higher in exponentially growing
promastigotes than in promastigotes stationary phase and amastigotes (M. Sousa Silva, data not
published). The author suggests that, during amastigote phase, the parasite requires an increase in
reduced thiols and anti-oxidative defences, which are NADPH-dependent. Therefore, in this life
cycle stage, the aldose reductase activity is lowered to decrease NADPH consumption, and the
glyoxalase enzymes’ activity antagonistically increased in order to keep methylglyoxal
concentration at low levels (M. Sousa Silva, data not published). The opposite is observed in
exponentially growing promastigotes. Considering the latter compensation system, it should be
required the inhibition of both glyoxalase and aldose reductase pathways, targeting the enzymes’
potentially different substrate binding sites relatively to the respective human counterparts, in order
to achieve an effective increase of methylglyoxal concentration with harmful effects to the parasite.
In fact, metabolic pathways, such as glycolysis (El Fakhry et al. 2002) and trypanothione-dependent
pathways (M. Sousa Silva, data not published), seem to be efficiently regulated in L. infantum.
The integrative study of both glyoxalase enzymes and aldose reductase performed
throughout the present work, is a step further in the structural and biochemical knowledge of both
L. infantum in general and trypanothione specificity. Although none of these enzymes might be
considered as attractive drug targets per se the three enzymes can be synergistically exploited in the
hunt for new anti-leishmanial drug targets.
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