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Catalytic Site Atlas

CSA LITERATURE entry for 1myr

E.C. namethioglucosidase
SpeciesSinapis alba (Brassica hirta)
E.C. Number (IntEnz) 3.2.1.147
CSA Homologues of 1myrThere are 27 Homologs
CSA Entries With UniProtID P29736
CSA Entries With EC Number 3.2.1.147
PDBe Entry 1myr
PDBSum Entry 1myr
MACiE Entry 1myr

Literature Report

IntroductionMyronase is the enzyme resposible for the hydrolysis of a variety of plant anionic-1-thio-beta-glucosides, called glucosinolates. Sinapsis alba myrosinase is a 499 amino acid S-glucosidase which hydrolyses an S-glycosidic bond, and is closely related to the O-glucosidases. Upon hydrolysis, configuration at the anomeric centre is retained.
The protein fold into a (beta/alpha)8 barrel structure, forming a dimer which is stabilised by a Zn ion, and is heavily glycosylated.
Myrosinases are thought to be involved in defence systems developed by plants
MechansimThe catalytic mechanism is a two-step process: a) The glycosylation step- formation of the gycosyl-enzyme complex with aglycon departure. b) Deglycosylation - hydrolysis of the glycosyl-enzyme complex by a nucleophilic water molecule.
1. The substrate binds to the enzyme in the hydrophobic pocket by glucosinolate hydrophobic side chains, and with two arginine residues positioned appropriately for interaction with the substrate sulphate group. This is via nucleophilic attack of Glu 409 on the substrate anomeric carbon, with aglycon departure. Ser 190 defines Glu 409 position and stabilises the glycosyl-enzyme complex by hydrogen bonding to the substrate sulphate group. Gln 187 activates a water molecule to assist acglycon departure, rather than acting as an acidic residue itself. 2. The sugar ring is bound to the enzyme via an alpha-glycosidic linkage to the Glu 409 residue, the catalytic nucleophile. 3. Upon formation of the glycosyl-enzyme complex, the salt bridge between Glu 409 and Arg 95 is disrupted, and the side chain of Glu 409 aletrs its conformation. 4. A water molecule hydrogen bonds to the Arg 95, whose charge becomes buried. 5. Formation of the glycosyl-enzyme complex is also accompanied by the change in position of Tyr 330, whose hydroxyl group points towards the sugar ring O. 6. hydrolysis of the glycosyl-enzyme intermediate by water. During the earlier nucleophilic attack, the water molecule moves closer to the C1 of the gly-enzyme complex by hydrogen bonding to the Oe1 of Gln 187. The Gln 187 position is defined by hydrogen bonds between Ser 190 gamma-O and Ne2 of Gln 187, and also between N-delta -2 of Asn 328 with Oe1 of Gln 187. This ensures precise positioning of the nucleophilic water molecule that is required for hydrolysis.
Reaction

Catalytic Sites for 1myr

Annotated By Reference To The Literature - Site 3 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GlnA187187macie:sideChainGln 187 is involved in hydrogen bonding to the nucleophilic water, which attacks the scissile bond in the deglycosylation step. This ensures specific positioning of the water molecule for nuclephilic attack.
AsnA328328macie:sideChainAsn 328 hydrogen bonds to the Oe1 of Gln 187 through its N-delta-2, which indirectly ensures the required positioning of the nucleophilic water molecule.
ArgA9595macie:sideChainArg 95 forms a salt bridge the the catalytic Glu 409, which is disrupted on formation of the gly-enzyme complex. This is stabilised by hydrogen bonding of Arg 95 to a neighbouring water molecule, so that the charge of the residue is buried
GluA409409macie:sideChain Glu 409 is the catalytic nucleophile and attacks the anomeric carbon of the sugar substrate to form the gly-enzyme complex. Specific positioning of Glu 409 is required and this is achieved by the disruption of the salt bridge between Glu 409 and Arg 95, so that the Glu 409 can change its conformation favourably
TyrA330330macie:sideChainTyr 330 stabilises the transition state by changing position so that its hydroxyl group points towards the sugar ring O.
SerA190190macie:sideChainer 190 hydrogen bonds to the Ne2 of Gln 187 through its gamma-O, which indirectly ensures the required positioning of the nucleophilic water molecule

Literature References

Notes:Based on modelling, the Tyr 180 hydroxyl group may be in proximity of the sulphur of the thioglycosidic linkage, suggesting a possible catalytic role for this residue.
Burmeister WP
The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase.
Structure 1997 5 663-675
PubMed: 9195886
Husebye H
Crystal structure at 1.1 Angstroms resolution of an insect myrosinase from Brevicoryne brassicae shows its close relationship to beta-glucosidases.
Insect Biochem Mol Biol 2005 35 1311-1320
PubMed: 16291087
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