4EWL

Crystal Structure of MshB with glycerol and Acetate bound in the active site

Summary for 4EWL

• Entry DOI: 10.2210/pdb4ewl/pdb
• Descriptor: 1D-myo-inositol 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase, 2-{2-[2-(2-{2-[2-(2-ETHOXY-ETHOXY)-ETHOXY]-ETHOXY}-ETHOXY)-ETHOXY]-ETHOXY}-ETHANOL, GLYCEROL, ... (6 entities in total)
• Functional Keywords: active site, glycerol, acetate, tyrosine 142, histidine 144, mshb, hydrolase
• Biological source: Mycobacterium tuberculosis
• Total number of polymer chains: 2
• Total formula weight: 64708.46

Authors

Broadley, S.G.,Sewell, B.T.,Weber, B.W.,Marakalala, M.J.,Steenkamp, D.J. (deposition date: 2012-04-27, release date: 2012-09-26, Last modification date: 2023-09-13)

Primary citation

Broadley, S.G.,Gumbart, J.C.,Weber, B.W.,Marakalala, M.J.,Steenkamp, D.J.,Sewell, B.T.
A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism.
Acta Crystallogr.,Sect.D, 68:1450-1459, 2012

PubMed Abstract: MshB, a zinc-based deacetylase, catalyses a step in the mycothiol biosynthetic pathway that involves the deacetylation of 1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcN-Ins) and acetate. In this study, MshB was expressed, purified and crystallized. A new crystal form was encountered in 0.1 M sodium acetate, 0.2 M ammonium sulfate, 25% PEG 4000 pH 4.6. The crystals diffracted to 1.95 Å resolution and the resulting electron-density map revealed glycerol and the reaction product, acetate, in the active site. These ligands enabled the natural substrate GlcNAc-Ins to be modelled in the active site with some certainty. One acetate O atom is hydrogen bonded to Tyr142 and is located 2.5 Å from the catalytic zinc. The other acetate O atom is located 2.7 Å from a carboxylate O atom of Asp15. This configuration strongly suggests that Asp15 acts both as a general base catalyst in the nucleophilic attack of water on the amide carbonyl C atom and in its protonated form acts as a general acid to protonate the amide N atom. The configuration of Tyr142 differs from that observed previously in crystal structures of MshB (PDB entries 1q74 and 1q7t) and its location provides direct structural support for recently published biochemical and mutational studies suggesting that this residue is involved in a conformational change on substrate binding and contributes to the oxyanion hole that stabilizes the tetrahedral intermediate.

PubMed: 23090394
DOI: 10.1107/S090744491203449X

Experimental method

X-RAY DIFFRACTION (1.85 Å)