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Glycosidases and Glycosyltransferases

•  Introduction to Inverting/Retaining Mechanisms

•  Inhibitor design

•  Chemical Reaction •  Proposed catalytic mechanisms

Multiple slides courtesy of Harry Gilbert with Wells modifications

Glycosidic bond cleavage

•  Classic example is lysozyme: cleaves N-acetlymuramic acid-β-4-GlcNAc

•  Discovered by Alexander Fleming in 1920s –  Sneezed onto his bacterial agar plate –  Bacteria found to be lysed next day –  Potential antimicrobial enzyme –  He discovered a better antimicrobial agent later; what is it?

Glycone Aglycone H2O

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Glycosidic bond cleavage in free solution

Glycone Aglycone H2O

Transition state oxocarbenium ion attacked by hydroxyl ion

Rate of glycosidic bond cleavage

•  The transition state (positively charged oxocarbenium ion) is a very high energy molecule –  Geometry changes from

chair to half-chair –  Why? –  So C1 and ring oxygen are

in same plane –  So positive charge is not

just at C1 but shared between C1 and ring oxygen

–  This stabilises positive charge.

–  Need lots of energy to cause change in geometry of sugar

C1

O5

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Two different mechanisms of acid-base assisted catalysis

•  Single displacement mechanism –  Inversion of the anomeric configuration of glycone

sugar

β-glycosidic bond Bond is equatorial

sugar OH is axial

Two different mechanisms of acid-base assisted catalysis

•  Double displacement mechanism –  Retention of the anomeric configuration of glycone

sugar

β-glycosidic bond Bond is equatorial

OH remains equatorial

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Two different mechanisms of acid-base assisted catalysis

•  How does an enzyme generate protons and hydroxyl ions?

•  Two amino acids with carboxylic acid side-chains –  Glutamate or aspartate

•  Two mechanisms are as follows:

Acid-base assisted single displacement mechanism

•  The acid catalyst –  Uncharged –  Hydrogen in the perfect position to be donated to the glycosidic

oxygen. •  The catalytic base

–  Extracts a proton from water –  Hydroxyl ion in the perfect position to attack C1 of the transition

state

Catalytic base

Catalytic acid

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Acid-base assisted double displacement mechanism

•  Two distinct reactions –  Glycosylation

•  Formation of a covalent glycosyl-enzyme intermediate (ester bond) •  The aglycone sugar released from active site

–  Deglycosylation •  The ester bond between the glycone sugar and the enzyme is hydrolysed

and the glycone sugar is released from the active site

Catalytic acid-base

Catalytic nucleophile

•  The first enzyme structure solved

•  The textbook example of enzyme catalyzed glycoside hydrolysis

•  Hydrolyses the glycosidic bond via a retaining mechanism

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Asp52

And the lysozyme mechanism is revisited: Covalent enzyme intermediate for hen egg

white lysozyme

Lysozyme (E35Q)

Vocadlo et al. Nature 412, 835-8

How can we identify the catalytic amino acids

•  Glycoside hydrolases are grouped in enzyme families based on sequence similarity (i.e. evolved from a common ancestor. Currently 100+ families –  http://afmb.cnrs-mrs.fr/CAZY/

•  All members of same family have –  Evolved from the same progenitor sequence –  Conserved mechanism –  Same fold –  Conserved catalytic apparatus

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CAZY

•  Several families have ancient ancestral relationship

•  Same fold, mechanism and catalytic residues

•  How does CAZY help us? •  Tells us what the catalytic residues are •  Tells us the mechanism •  Tells us the likely substrate specificity

Sequence 1:73 QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN 1 UNIPROT:XYNA_PSEFL 1:73 335:407 QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN 2 UNIPROT:Q9AJR9 1:68 111:178 RHNQQVRGHNLCWHE--ELPTwaSEVngNAKEILIQHIQTVAGRYAGRIQSWDVVNEAILPKDGRPDG----- 3 UNIPROT:GUX_CELFI 3:66 115:176 --GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFADG-DGP------- 4 UNIPROT:Q59277 3:61 116:173 --GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFAD------------ 5 UNIPROT:Q59675 1:63 324:391 ENNMTVHGHALVWHSDYQVPnwAGSAE-DFLAALDTHITTIVDHYegNLVSWDVVNEAIDDNS---------- 6 UNIPROT:Q59301 2:63 343:409 -NNINVHGHALVWHSDYQVPNFmsGSAADFIAEVEDHVTQVVTHFkgNVVSWDVVNEAINDGS---------- 7 UNIPROT:Q59139 1:73 111:180 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMAHYKGKIVQWDVVNEAFADG--NSGGRRDSN 8 UNIPROT:Q7SI98 1:73 73:142 QNGKQVRGHTLAWHS--QQPGWMQssGSTLRQAMIDHINGVMGHYKGKIAQWDVVNEAFSD--DGSGGRRDSN 9 UNIPROT:XYNB_THENE 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS----------- 10 UNIPROT:Q60044 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS----------- 11 UNIPROT:AAN16480 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS----------- 12 UNIPROT:Q7TM36 8:68 2:58 -------GHTVVWHGA--VPTWLNasTDDFRAAFENHIRTVADHFRGKVLAWDVVNEAV---ADDGSG----- 13 UNIPROT:Q7WVV0 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS----------- 14 UNIPROT:Q7WUM6 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS----------- 15 UNIPROT:Q9WXS5 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS----------- 16 UNIPROT:Q9P973 1:57 120:176 QNGKSIRGHTLIWHS--QLPAWVNnnNAdlRQVIRTHVSTVVGRYKGKIRAWDVVNE---------------- 17 UNIPROT:Q9X584 1:63 115:176 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIAQWDVVNEAFADGS---------- 18 UNIPROT:XYNA_STRLI 1:63 114:175 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS---------- 19 UNIPROT:Q8CJQ1 1:63 114:175 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS---------- 20 UNIPROT:P79046 1:62 93:155 QNGQGLRCHTLIWYS--QLPGWVSSGNWN-RQTLEahIDNVMGHYKGQCYAWDVVNEAVDDN----------- 21 UNIPROT:Q9XDV5 3:71 427:505 --GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS-- 22 UNIPROT:Q8GJ37 3:71 427:505 --GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS-- 23 UNIPROT:Q7X2C9 1:63 27:88 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMNHSKGKIAQWDVVNEAFADGS---------- 24 UNIPROT:Q9RJ91 3:61 105:162 --GMDVRGHTLVWHS--QLPSWVSPLGadLRTAMNAHINGLMGHYKGEIHSWDVVNEAFQD------------ 25 UNIPROT:Q59922 3:61 119:176 --GMKVRGHTLVWHS--QLPGWVSPLAadLRSAMNNHITQVMTHYKGKIHSWDVVNEAFQD------------ 26 UNIPROT:Q9RMM5 1:61 113:172 QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED------------ 27 UNIPROT:BAD02382 1:61 113:172 QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED------------

Catalytic acid

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Inhibitors of glycoside hydrolases

•  Glycoside hydrolase activities contribute to significant diseases – Flu – Type II diabetes – Possibly Cancer and Aids

•  To combat diseases need to develop inhibitors

Designing glycoside hydrolase inhibitors

•  What comprises a good inhibitor? •  Mechanistic covalent inhibitors not used •  Very high affinity non-covalent

competitive inhibitors – Transition state inhibitors

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deglycosylation

glycosylation

Transition state has a positive charged nature as leaving group departure precedes nucleophile attack

TS-based inhibitors that mimic charge distribution

deoxynojirimycin

isofagamine

Both have nM Ki values. Affinities are about one million times higher than substrate

Why are they transition state mimics?

Contains a positive charge

Glucosidase Inhibitors

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Mimicking the half-chair •  Insert a double-bond to enforce planarity

Drugs that mainly mimic the half chair All picomolar affinities 108-fold tighter binders

than substrates HIV drug: prevents glycosylation in mammalian cells AIDs virus surface proteins are not glycosylated and thus can’t evade the immune system Type II diabetes

(inhibits human Amylase)

Anti-flu drugs

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Annual Reviews

•  Two folds •  Both have two Rossman domains •  GTA strongly linked may look like a single β-

sheet •  GT-B has two separate domains •  Requirement of nucleotide binding limits number

of folds greatly

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Inverting GT

Retaining GT

Inverting GT

Retaining GT

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Take Home Points

•  CAZY •  Inverting/Retaining Mechanisms •  Mechanistic Based Inhibitors

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References •  Cantarel et al (2008) Nucleic Acid Res 37:D233-8

(CAZY) •  Vocadlo at al. (2001) Nature 412:835-8. (Mechanistic

inhibitors of glycoside hydrolases) •  Lairson et al. (2008) Ann. Rev. Biochem. 77:521-555

(glycosyltransferases) •  Rye and Withers (2000) Curr. Opin. Chem. Biol.

4:573-580 (glycoside hydrolases) •  Tailford (2008) Nature Chem. Biol. Nat. 4:306-12

(Transition state geometry)

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3 6 Ser/Thr

β3

Ser/Thr

β4

β3

β4

β3

β4

α3

α3

α3

Ser/Thr

β3 β6

Asn

Asn

AsnAsn

Asn

2 2 64 4

Ser/Thr

Ser/Thr

A. B. C. D.

G. H. I. J. K.

E. F.

Asn