Publications

• The substrate-induced conformational change of Mycobacterium tuberculosis mycothiol synthase.

  Vetting M.W., Yu M., Rendle P.M., Blanchard J.S.

  The structure of the ternary complex of mycothiol synthase from Mycobacterium tuberculosis with bound desacetylmycothiol and CoA was determined to 1.8 A resolution. The structure of the acetyl-CoA-binary complex had shown an active site groove that was several times larger than its substrate. The ... >> More

  The structure of the ternary complex of mycothiol synthase from Mycobacterium tuberculosis with bound desacetylmycothiol and CoA was determined to 1.8 A resolution. The structure of the acetyl-CoA-binary complex had shown an active site groove that was several times larger than its substrate. The structure of the ternary complex reveals that mycothiol synthase undergoes a large conformational change in which the two acetyltransferase domains are brought together through shared interactions with the functional groups of desacetylmycothiol, thereby decreasing the size of this large central groove. A comparison of the binary and ternary structures illustrates many of the features that promote catalysis. Desacetylmycothiol is positioned with its primary amine in close proximity and in the proper orientation for direct nucleophilic attack on the si-face of the acetyl group of acetyl-CoA. Glu-234 and Tyr-294 are positioned to act as a general base and general acid to promote acetyl transfer. In addition, this structure provides further evidence that the N-terminal acetyltransferase domain no longer has enzymatic activity and is vestigial in nature. << Less

  J. Biol. Chem. 281:2795-2802(2006) [PubMed] [EuropePMC]

• Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases.

  Vetting M.W., Roderick S.L., Yu M., Blanchard J.S.

  Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys ... >> More

  Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol. << Less

  Protein Sci. 12:1954-1959(2003) [PubMed] [EuropePMC]

• Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes.

  Koledin T., Newton G.L., Fahey R.C.

  Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed agai ... >> More

  Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2-L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene. << Less

  Arch. Microbiol. 178:331-337(2002) [PubMed] [EuropePMC]