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

ABSTRACT: 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.

SUBMITTER: Tremblay LW

PROVIDER: S-EPMC2628295 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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

Tremblay L W LW Fan F F Vetting M W MW Blanchard J S JS

Biochemistry 20081201 50

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 struc ...[more]

PMID: 19053270

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Project description:Mycobacterium tuberculosis and other members of the actinomycete family produce mycothiol (MSH or acetylcysteine-glucosamine-inositol, AcCys-GlcN-Ins) to protect the organism against oxidative and antibiotic stress. The biosynthesis of MSH proceeds via a five-step process that involves four unique enzymes, MshA-D, which represent specific targets for inhibitor design. Recombinant Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of glucosamine-inositol (GlcN-Ins) and cysteine to form Cys-GlcN-Ins. The 1.6 A three-dimensional structure of MshC in complex with a tight binding bisubstrate analogue, 5'-O-[N-(L-cysteinyl)sulfamonyl]adenosine (CSA), has suggested specific roles for T46, H55, T83, W227, and D251. In addition, a catalytic role for H55 has been proposed on the basis of studies of related aminoacyl-tRNA synthetases. Site-directed mutagenesis was conducted to evaluate the functional roles of these highly conserved residues. All mutants exhibited significantly decreased k(cat) values, with the exception of T83V for which a <7-fold decrease was observed compared to that of the wild type (WT). For the T46V, H55A, W227F, and D251N mutants, the rate of cysteine activation decreased 100-1400-fold compared to that of WT, consistent with the important roles of these residues in the first half-reaction. The approximately 2000-fold decrease in k(cat)/K(m) as well as the approximately 20-fold decrease in K(m) for cysteine suggested a significant role for T46 in cysteine binding. Kinetic studies also indicate a function for W227 in cysteine binding but not in substrate discrimination against serine. H55 was also observed to play a significant role in ATP binding as well as cysteine adenylation. The activity of H55A was partially rescued with exogenous imidazole at acidic pH values, suggesting that the protonated form of histidine is exerting a catalytic role. The pH dependence of the kinetic parameters with the WT enzyme suggests an additional requirement for a catalytic base in cysteinyl ligation.

Dataset Information

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

Publications

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

Similar Datasets

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