PDBsum entry 1e4m

Hydrolase

PDB id: 1e4m

Contents

Protein chain

499 a.a. *

Ligands

NAG-NAG

NAG-NAG-BMA-XYP-FUC

NAG-NAG-BMA-XYP-MAN-MAN-FUC

NAG ×6

SO4 ×8

GOL ×5

Metals

_ZN

Waters ×797

* Residue conservation analysis

PDB id:

1e4m

Name:

Hydrolase

Title:

Myrosinase from sinapis alba

Structure:

Myrosinase ma1. Chain: m. Synonym: sinigrinase, thioglucoside, glucohydrolase. Ec: 3.2.1.147

Source:

Sinapis alba. White mustard. Organism_taxid: 3728. Strain: emergo. Organ: seed. Cellular_location: myrosin grains

Biol. unit:

Dimer (from PDB file)

Resolution:

1.20Å R-factor: 0.124 R-free: 0.142

Authors:

W.P.Burmeister

Key ref:

W.P.Burmeister et al. (2000). High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. J Biol Chem, 275, 39385-39393. PubMed id: 10978344 DOI: 10.1074/jbc.M006796200

Date:

10-Jul-00 Release date: 25-May-01

Protein chain

P29736  (MYRA_SINAL) -  Myrosinase MA1 from Sinapis alba

Seq: 501 a.a.

Struc: 499 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.147 - thioglucosidase.
• Reaction: a thioglucoside + H2O = a sugar + a thiol

thioglucoside (Bound ligand (Het Group name = BMA) matches with 84.62% similarity) + H2O = sugar + thiol

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M006796200

J Biol Chem 275:39385-39393 (2000)

PubMed id: 10978344

High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base.

W.P.Burmeister, S.Cottaz, P.Rollin, A.Vasella, B.Henrissat.

ABSTRACT

Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-beta-d-glucosides (glucosinolates) considered part of the plant defense system. Although O-glycosidases are ubiquitous, myrosinase is the only known S-glycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, d-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis, i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase.

Selected figure(s)

Figure 4.
Fig. 4. The binding of ascorbate. The structures have been obtained on crystals soaked with ascorbic acid and different inhibitors. Electron density maps as described for Fig. 2. Water molecules are shown as red spheres. The refined structures are shown, including ascorbate, water molecules, sulfate ions, glycerol, inhibitors, and active site residues. Hydrogen bonds involved in ascorbate recognition are shown as dotted lines. a, soak with ascorbate, the glycerol molecule comes from the cryoprotectant. b, ascorbate and gluco-hydroximolactam. The ascorbate competes with the sulfate ion that has both partial occupancies of 0.6 for the ascorbate and 0.4 for the sulfate. c, ascorbate bound to the 2-F-glucosyl enzyme. The water molecule that is activated by the ascorbate for an attack on the C-1 carbon of the glucose is indicated by a pink arrow.

Figure 8.
Fig. 8. Schematic reaction mechanism of myrosinase in the absence (a) and presence (b and c) of ascorbic acid. E, enzyme; S, substrate; GE, glucosyl enzyme; A, ascorbate; k[1], k[2], etc., dissociation constants not involving ascorbate; k[3'], rate constant in presence of ascorbate; k[A1], k[A 1], etc., dissociation constants of ascorbate. The less important back reactions are not shown.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 39385-39393) copyright 2000.

Figures were selected by the author.

Author's comment

The enzyme is a dimer linked in particular through a Zn2+ ion (ZN1502) located on the 2-fold dimer axis and coordinated by 2 aspartic acid (ASP M70)and 2 histidine residues (HIS M56). The full heptasaccharide typical for plant glycosylation is visible attached to ASN M292.
Wim Burmeister