Browsing by Author "Broadley, Simon Gareth"

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    A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism.
    (International Union of Crystallography, 2012) Broadley, Simon Gareth; Gumbart, James Conrad; Weber, Brandon William; Marakalala, Mohlopheni Jackson; Steenkamp, Daniel Jacobus; Sewell, Bryan Trevor
    MshB, a zinc-based deacetylase, catalyses a step in the mycothiol biosynthetic pathway that involves the deacetylation of 1-O-(2-acetamido-2-deoxy--d-glucopyranosyl)-dmyo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy--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 A˚ 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 A˚ from the catalytic zinc. The other acetate O atom is located 2.7 A˚ 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.
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    Substrate binding to MshB, a zinc-dependent deacetylase in the mycothiol biosynthetic pathway of Mycobacterium tuberculosis
    (2012) Broadley, Simon Gareth; Sewell, Bryan Trevor
    New drugs are needed to combat the ever-increasing incidence of drug resistant tuberculosis. Eukaryotes and Gram negative bacteria make use of a low molecular weight thiol-containing peptide, glutathione (GSH), in their defense against toxins and oxidative stress. In mycobacteria a carbohydrate-based thiol, mycothiol (MSH), is used instead. MshB catalyses the third step in the mycothiol biosynthetic pathway which involves the deacetylation of 1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcNAcIns), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myoinositol (GlcN-Ins) and acetate. MshB is a homolog of another zinc-dependent amidase, mycothiol S-conjugate amidase (Mca), which recycles GlcNAc-Ins by cleaving the similarly located amide bond after mycothiol has reacted with an electrophile. The enzymes have overlapping enzyme activity and their products feed into the same step of the mycothiol synthetic pathway. Structure-based drug design could potentially be used to develop leads that could inhibit both enzymes simultaneously. This study was motivated by the need to visualise a putative inhibitor bound to MshB. An attempt was made to co-crystallise VU5 and MshB. VU5, which comprises plumbagin tethered to a thiophenylglycoside by five methylene carbons, was found by Gammon et al. (2010) to be an inhibitor of MshB and Mca. Diffraction quality crystals were grown and data were collected at the ESRF synchrotron facility. The crystal structure was solved by molecular replacement and most notably, glycerol and acetate (a product of the natural reaction) was found bound in the active site. This allowed a greater understanding regarding important residues involved in substrate binding and the catalytic mechanism. The findings presented here provide some insight into the substrate binding mechanism and support a recent publication that infers that Tyr-142 plays an important role in the catalytic mechanism, as well as suggesting that VU5 may behave as a substrate and that DTT may act as an inhibitor.