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The Biosynthesis of Alliin
Jill Maria HughesJill Maria Hughes
EU Project No.QLK1-CT-1999-00498
with Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett and Angela
Tregova
Biochemistry of Garlic at Liverpool
How is Alliin synthesisedHow is Alliin synthesised
Where is it synthesised?When is it synthesised?
Biosynthetic PathwaySO4
2- SO32- SO2
2- cysteine
glutathione(γ-glu-cys-gly)
S-methyl-γ-glu-cys
gly
S-methylcysteine
methiin
glu
trans-peptidase
oxidase
S-2-CP-γ-glu-cys
gly
S-trans-1-propenyl-γ-glu-cys
S-trans-1-propenylcysteineoxidase
trans-peptidase
glu
HCOOH
S-trans-1-propenylcysteine sulphoxide(isoalliin)
S-methylglutathioneS-(2-carboxypropyl)-glutathioneS-allylglutathione
S-allyl-γ-glu-cys
gly
S-allylcysteine
glu
trans-peptidase
oxidase
alliin
Allyl group(unknown sources)
valine & methacrylateserine
oxidase
S-allylcysteine
S-allyl-cysteine sulphoxide(alliin)
The Serine and Glutathione Pathways
Allyl Cysteine
Alliin
Serine pathway
SerineUnknown Allyl source
Cysteine
Allyl Glutathione
Gamma-Glutamyl Allyl Cysteine
Glutathione pathway
Glutathione
Our Approach this year
Precursor feeding. Precursor feeding. Modify method from isocratic to gradient.Modify method from isocratic to gradient.Use HPLC to identify intermediates.Use HPLC to identify intermediates.
Allyl Cysteine Synthase.Allyl Cysteine Synthase.Identification of a specific garlic enzyme that can Identification of a specific garlic enzyme that can
synthesise Allyl Cysteine.synthesise Allyl Cysteine.
HPLC Method Development
•Heptane sulphonic acid method was tried but was not suitable for indentification of compounds by Mass Spectroscopy
•We developed a simple HCl:Acetonitrile gradient which allows simultaneous identification of cysteine sulphoxides and gamma-glutamyl peptides.
•This method is now in routine use in our laboratory.
To aid identification of cysteine conjugates, our simple isocratic HPLC Method has been adapted:
HPLC Method Development
0.00 10.00 20.00 30.00 40.00
0
20
40
60
80
100
Alliin
Isoalliin
Gamma-glutamylallylcysteine
Typical HPLC trace of Garlic clove
gradient
HPLC Method Development
•Compare Compare unknown peak unknown peak retention times retention times with standardswith standards
•Mass spectroscopy-identify from ‘general profile’ and mass of molecular ion
Standards Rt 0.9ml/min
Methyl CSO 4.25Ethyl CSO 3.76Methyl cysteine 4.25Alliin 4.49Propenyl CSO 5.24Propyl CSO 5.76Glutathione 6.01Gamma glutamyl cysteine 6.78Methionine 6.74Ethyl cysteine 7.04Methyl glutathione 10.113-CPC 10.562-CPC 11.74Allyl cysteine 11.69Butyl CSO 12.061,2 Dicarboxy glutathione 12.53Propenyl cysteine 14.66propyl cysteine 15.44Gamma glutamyl allyl cysteine 20.19Butyl cysteine 21.86Propyl glutathione 20.8
Synergi RPMax 100%0.03MHCl@5min 50%Acetonitrile@25min
Peak Identification:Peak Identification:
HPLC Method Development
0.00 10.00 20.00 30.00 40.00
0
20
40
60
80
100
Alliin
Isoalliin
Gamma-glutamylallylcysteine
Gamma-glutamylisoallylcysteine
Typical HPLC trace of Garlic clove
gradient
Precursor Feeding ExperimentsGAt2b GPt2a GAC2b GPC2c Av GBl2
Alliin 41 42 509 6 14Propiin 12 135 14 26 3Allyl cysteine 81 0 198 0 0Propyl cysteine 5 85 12 138 0
GAt6b GPt6c GAC6a GPC6a Av GBl6Alliin 0 56 22 24 20Propiin 0 239 14 61 4Allyl cysteine 36 34 348 27 0Propyl cysteine 0 161 8 248 6
GAt13a GPt13b GAC13b GPC13b Av GBl13Alliin 0 23 53 30 14Propiin 0 203 6 167 12Allyl cysteine 0 0 302 0 0Propyl cysteine 0 60 0 254 11
OnAt2c OnPt2a OnAC2a OnPC2b Av OnBl2Alliin 146 12 172 80 77Propiin 9 31 27 30 10Allyl cysteine 196 0 446 0 0Propyl cysteine 23 80 25 170 14
OnAt6c OnPt6a OnAC6a OnPC6b Av OnBl6Alliin 131 74 586 109 88Propiin 24 103 9 325 13Allyl cysteine 286 65 716 0 0Propyl cysteine 23 163 0 278 30
OnAt13c OnPt13b OnAC13b OnPC13b Av OnBl13Alliin 34 12 348 57 53Propiin 12 106 11 171 16Allyl cysteine 153 0 349 0 0Propyl cysteine 9 21 8 188 15
Onion
Garlic
In Allium callus tissue the general reaction seems to apply:
Alk(en)yl thiol
Alk(en)yl cysteine
Alk(en)yl CSO
Using single clone garlic callus to reduce variation
Precursor Feeding Experiments-
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0
20
40
60
80
100
0.00 10.00 20.00 30.00 40.00
0
20
40
60
80
100
0.00 10.00 20.00 30.00 40.00
0
20
40
60
80
100
Onion callus incubated with allyl thiol and propyl thiol for six days.
Allyl thiol
Propyl thiol
AlliinAllyl Cysteine
Propiin Propyl cysteine
Both Garlic and Onion callus can convert exogenously applied allyl thiol and propyl thiol to their respective cysteine conjugate and subsequently to their respective cysteine sulphoxide(CSO)
Control
Is there an Allyl Cysteine Synthase?
Some purified plant cysteine synthase enzymes have shown the capability of producing cysteine conjugates in addition to their normal function
Is there a specific cysteine synthase homologue in garlic that can conjugate allyl thiol to (O-acetyl)serine to give allyl cysteine and subsequently alliin?
Sulphide + O-Acetyl Serine Cysteine
Allyl thiol + O-Acetyl Serine Allyl Cysteine Alliin
Cysteine synthase
Allyl Cysteine synthase
The Serine and Glutathione Pathways
Allyl Cysteine
Alliin
Serine pathway
Serine Unknown Allyl source
Cysteine
Allyl Glutathione
Glutathione pathway
Glutathione
Allyl Cysteine synthase Gamma-
Glutamyl Allyl Cysteine
Cysteine synthase activity. Q-Sepharose. 7.11.01
00.05
0.10.15
0.20.25
0.3
Fraction
OD
cysteine synthaseactivity
Many fractions show cysteine synthase activity
Protein purification:Ion Exchange chromatography
Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography.
Only a few fractions show allyl cysteine synthase activity
Protein purification: Hydrophobic Interaction Chromatography
Allyl cysteine synthase and cysteine synthase activity co-elute
Phenyl sepharose fractionation
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 5 7 9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
Fraction
OD
55
0
cysteinesyntase activity
allyl cysteinesynthaseactivity
Cysteine production was assayed colorimetrically and allyl cysteine by HPLC
Protein purification: How pure is the Fraction?
SDS-PAGE shows a distinct band in the allyl cysteine synthase active fractions at approx. 34000 kdal.
This M Wt. is consistent with plant cysteine synthase monomers found previously
Fractions 26 27 28 29 30
34000
What is the Enzyme?
The selected 34000 band was digested with trypsin and the resultant peptides separated by preparative HPLC.
Three selected peptides were sequenced:-
…….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP………….
A simple BLAST search of these peptides in the protein database shows most similarity to a cysteine synthase from Oryza sativa (Rice)
Biochemistry of Garlic at Liverpool
Where now?
This enzyme will be selected and overexpressed
Enyme kinetics of the purified enzyme should help to clarify it’s role in vivo.
The future?
Use similar techniques to probe the glutathione pathway?
The Serine and Glutathione Pathways
Allyl Cysteine
Alliin
Serine pathway
Serine Unknown Allyl source
Cysteine
Allyl Glutathione
Gamma- Glutamyl Allyl Cysteine
Glutathione pathway
Glutathione
Allyl Cysteine synthase
Glutathione-S-Transferase
The Biosynthesis of Alliin
Jill Maria HughesJill Maria Hughes
Acknowledgements:
Mark Prescott Mass Spectroscopy
Mark Wilkinson Protein purification facilities and Peptide SequencingEU Project No.QLK1-CT-1999-00498