Acta Crystallographica Section D

Biological Crystallography

Volume 68, Part 11 (November 2012)

research papers

Acta Cryst. (2012). D68, 1450-1459    [ doi:10.1107/S090744491203449X ]

A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism

S. G. Broadley, J. C. Gumbart, B. W. Weber, M. J. Marakalala, D. J. Steenkamp and B. T. Sewell

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-[alpha]-D-glucopyranosyl)-D-myo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy-[alpha]-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.

PDB reference: 4ewl

Keywords: MshB; Mycobacterium tuberculosis; catalytic mechanism; oxyanion hole; conformational change; hydrolases.

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Joint Photographic Experts Group (JPEG) image (689.3 kbytes)
[ doi:10.1107/S090744491203449X/sx5106sup1.jpg ]
Picture of the crystals.

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Protein Data Bank file (355.4 kbytes)
[ doi:10.1107/S090744491203449X/sx5106sup2.pdb ]
Coordinates of a time point in the molecular-dynamics-derived model of MshB with the natural substrate GlcNAc-Ins bound in the active site, as shown in Fig. 3 of the main article.

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Portable Document Format (PDF) file (372.2 kbytes)
[ doi:10.1107/S090744491203449X/sx5106sup3.pdf ]
Description of the locations of the two bound polyethylene glycol molecules.


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