The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis (
            <i>mshA</i>
            )

Newton, Gerald L.; Koledin, Teresa; Gorovitz, Batia; Rawat, Mamta; Fahey, Robert C.; Av‐Gay, Yossef

doi:10.1128/jb.185.11.3476-3479.2003

Cited by 78 publications

(93 citation statements)

References 22 publications

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“…2). The first dedicated intermediate in MSH biosynthesis is 1-O-(2-acetamido-2-deoxy-␣-D-glucopyranosyl)-Dmyo-inositol-3-phosphate (GlcNAc-Ins-P), produced from UDP-GlcNAc and Ins-P by the glycosyltransferase MshA (91,92). The second step involves the dephosphorylation of GlcNAcIns-P, catalyzed by MshA2, which is encoded by a gene that is not yet identified.…”

Section: Msh Biosynthesismentioning

confidence: 99%

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

Newton

Buchmeier

Fahey

2008

Microbiol Mol Biol Rev

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316

293

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SUMMARY Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria. MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves production of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH production, whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by σB and σR in Streptomyces coelicolor. Identified enzymes of MSH metabolism include mycothione reductase (disulfide reductase; Mtr), the S-nitrosomycothiol reductase MscR, the MSH S-conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S-conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S-transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.

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Section: Msh Biosynthesismentioning

confidence: 99%

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

Newton

Buchmeier

Fahey

2008

Microbiol Mol Biol Rev

Self Cite

316

293

View full text Add to dashboard Cite

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“…1). The first enzyme, MshA, catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to 1-L-myo-inositol 1-phosphate (1-L-Ins-1-P) to produce 3-phospho-1-D-myo-inosityl-2-acetamido-2-deoxy-␣-D-glucopyranoside (GlcNAc-Ins-P) (7,8). A yet to be discovered phosphatase is proposed to dephosphorylate GlcNAc-Ins-P to produce GlcNAc-Ins (7).…”

mentioning

confidence: 99%

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

Vetting

Frantom

Blanchard

2008

Journal of Biological Chemistry

114

192

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The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-Lmyo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-L-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97°rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-Lmyo-inositol-1-phosphate. The structure highlights the residues important in binding of UDP-N-acetylglucosamine and 1-L-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the ␤-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-L-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.

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“…The enzymes involved in MSH biosynthesis therefore present potential as drug targets for anti-mycobacterial treatment. Observations from the M. smegmatis strains with deficient or absent functions of MshA-D indicate that MshC has perhaps the greatest potential as a drug target, since inhibiting MshC function results in strains with a higher susceptibility towards conventional antibiotic treatment (2,3,(10)(11)(12)(13). Despite its significance, only preliminary kinetic measurements have been reported for the M. smegmatis enzyme (20).…”

Section: Discussionmentioning

confidence: 99%

“…This compares quiet well to the k cat value of 3.15 s −1 obtained from steady state kinetic measurements. (11) Comparison with aminoacyl-tRNA synthetases Since MshC shares significant sequence homology and catalyzes a similar reaction to cysteinyltRNA synthetase, mechanistic studies for aminoacyl-tRNA synthetases provides intriguing comparisons with MshC. Aminoacyl-tRNA synthetases can be divided into two classes, which differ in their structural folds and rate limiting steps (29).…”

Section: Pre-steady State Kineticsmentioning

confidence: 99%

Steady-State and Pre-steady-State Kinetic Analysis of Mycobacterium smegmatis Cysteine Ligase (MshC)

et al. 2007

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Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth, and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-D. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady state and pre-steady state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form CysGlcN-Ins. Initial velocity and inhibition studies show that the steady state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PP i , binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady state kinetic parameters were determined to be: k cat equal to 3.15 s −1 , and K m values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analog, 5′-O-[N-(Lcysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and non-competitive inhibition versus cysteine, with an inhibition constant of ~306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid quench techniques, giving rates of ~9.4 s −1 and ~5.2 s −1 , respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC. KeywordsMycobacterium; mycothiol; cysteine ligase Mycothiol (MSH, acetyl-Cys-GlcN-Ins), a conjugate of N-acetylcysteine (AcCys) and , is the predominant low molecular weight thiol that protects actinomycetes against oxidative stress and cellular electrophilic toxins (1-4). Among actinomycetes, mycobacteria generate the highest intracellular levels of MSH (5). Studies have shown that Mycobacterium smegmatis mutants lacking MSH become more sensitive towards oxidizing agents, electrophiles, and antibiotics (1-3), indicating the critical role of MSH in the survival and pathogenicity of mycobacteria (1). The detoxification mechanism of electrophiles by MSH has been proposed (Scheme 1, path a) to involve the formation of a MSH S-conjugate of the electrophile (1). The conjugate *AUTHOR EMAIL ADDRESS: blanchar@aecom.yu.edu. CORRESPONDING AUTHOR FOOTNOTE. Phone: (718) Fax: (718) It has been proposed that MSH is synthesized via a series of enzymatic reactions (6-9), as illustrated in Scheme 1, path b. In brief, the process is initiated by an N-acetyl-glucosamine transferase (MshA) to generate 3-phospho-GlcNAc-Ins, which is subsequently dephosphorylated to form GlcNAc-Ins by an unknown phosphatase (9). GlcNAc-Ins is subsequently deacetylated by MshB. The resulting GlcN-Ins is ligated with cysteine in a reaction catalyzed by a cysteine ligase, MshC. ...

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The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis ( mshA )

Cited by 78 publications

References 22 publications

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

Steady-State and Pre-steady-State Kinetic Analysis of Mycobacterium smegmatis Cysteine Ligase (MshC)

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