Substrate binding to MshB, a zinc-dependent deacetylase in the mycothiol biosynthetic pathway of Mycobacterium tuberculosis
Master Thesis
2012
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University of Cape Town
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Abstract
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.
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Includes bibliographical references.
Reference:
Broadley, S. 2012. Substrate binding to MshB, a zinc-dependent deacetylase in the mycothiol biosynthetic pathway of Mycobacterium tuberculosis. University of Cape Town.