Publications

• ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases.

  Sareen D., Steffek M., Newton G.L., Fahey R.C.

  Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins ... >> More

  Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins) catalyzed by MshC to produce Cys-GlcN-Ins. The ligase activity was purified approximately 2400-fold from Mycobacterium smegmatis and two proteins of slightly different M(r) approximately 47000 were identified with MshC activity. The N-terminal sequence of the smaller protein revealed that it was coded by a gene in the databases for M. smegmatis and M. tuberculosis previously designated as cysS2. The larger protein was coded by the same gene in M. smegmatis but included an eight amino acid N-terminal extension involving a different start codon. The ligase was found to have K(m) values of 40 +/- 3 and 72 +/-9 microM for Cys and GlcN-Ins, respectively. The cysS2 gene was thought to encode a second cysteinyl-tRNA synthetase in addition to cysS but the present results indicate that cysS2 is actually the mshC gene encoding ATP-dependent Cys:GlcN-Ins ligase. << Less

  Biochemistry 41:6885-6890(2002) [PubMed] [EuropePMC]

• Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC).

  Fan F., Luxenburger A., Painter G.F., Blanchard J.S.

  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-alpha-d-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for ce ... >> More

  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-alpha-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-MshD. 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 Cys-GlcN-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 PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s-1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5'-O-[N-(l-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of approximately 306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of approximately 9.4 and approximately 5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC. << Less

  Biochemistry 46:11421-11429(2007) [PubMed] [EuropePMC]

• The 1.6 A crystal structure of Mycobacterium smegmatis MshC: the penultimate enzyme in the mycothiol biosynthetic pathway.

  Tremblay L.W., Fan F., Vetting M.W., Blanchard J.S.

  Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and l-cysteine to form l-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl ... >> More

  Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and l-cysteine to form l-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-O-[N-(l-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 A. The refined structure exhibited electron density corresponding to the entire 47 kDa MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions. << Less

  Biochemistry 47:13326-13335(2008) [PubMed] [EuropePMC]