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

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dc.contributor.authorBroadley, Simon Gareth
dc.contributor.authorGumbart, James Conrad
dc.contributor.authorWeber, Brandon William
dc.contributor.authorMarakalala, Mohlopheni Jackson
dc.contributor.authorSteenkamp, Daniel Jacobus
dc.contributor.authorSewell, Bryan Trevor
dc.date.accessioned2016-07-25T10:17:51Z
dc.date.available2016-07-25T10:17:51Z
dc.date.issued2012
dc.date.updated2016-07-12T13:55:35Z
dc.description.abstractMshB, 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.en_ZA
dc.identifierhttp://dx.doi.org/10.1107/S090744491203449X
dc.identifier.apacitationBroadley, S. G., Gumbart, J. C., Weber, B. W., Marakalala, M. J., Steenkamp, D. J., & Sewell, B. T. (2012). A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism. <i>Acta Crystallographica Section D Biological Crystallography</i>, http://hdl.handle.net/11427/20672en_ZA
dc.identifier.chicagocitationBroadley, Simon Gareth, James Conrad Gumbart, Brandon William Weber, Mohlopheni Jackson Marakalala, Daniel Jacobus Steenkamp, and Bryan Trevor Sewell "A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism." <i>Acta Crystallographica Section D Biological Crystallography</i> (2012) http://hdl.handle.net/11427/20672en_ZA
dc.identifier.citationBroadley, S. G., Gumbart, J. C., Weber, B. W., Marakalala, M. J., Steenkamp, D. J., & Sewell, B. T. (2012). A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism. Acta Crystallographica Section D: Biological Crystallography, 68(11), 1450-1459.en_ZA
dc.identifier.issn0907-4449en_ZA
dc.identifier.ris TY - Journal Article AU - Broadley, Simon Gareth AU - Gumbart, James Conrad AU - Weber, Brandon William AU - Marakalala, Mohlopheni Jackson AU - Steenkamp, Daniel Jacobus AU - Sewell, Bryan Trevor AB - 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. DA - 2012 DB - OpenUCT DP - University of Cape Town J1 - Acta Crystallographica Section D Biological Crystallography LK - https://open.uct.ac.za PB - University of Cape Town PY - 2012 SM - 0907-4449 T1 - A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism TI - A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism UR - http://hdl.handle.net/11427/20672 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/20672
dc.identifier.vancouvercitationBroadley SG, Gumbart JC, Weber BW, Marakalala MJ, Steenkamp DJ, Sewell BT. A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism. Acta Crystallographica Section D Biological Crystallography. 2012; http://hdl.handle.net/11427/20672.en_ZA
dc.languageengen_ZA
dc.publisherInternational Union of Crystallographyen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.sourceActa Crystallographica Section D Biological Crystallographyen_ZA
dc.source.urihttp://journals.iucr.org/d/
dc.titleA new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism.en_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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