The 1.6 Å 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 Lcysteine 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-hour 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 Å. The refined structure exhibited electron density corresponding to the entire 47 kDalton 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. KeywordsMycobacterium; mycothiol; cysteine ligase; limited proteolysis; three-dimensional structure Actinomycetes produce mycothiol (MSH, acetyl-cys-GlcN-Ins) as the predominant low molecular weight thiol to minimize oxidative stress and protect against electrophilic toxins (1-4). Among actinomycetes, mycobacteria generate the highest intracellular levels of MSH (5). Mycobacterium smegmatis mutants which are deficient in MSH production 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). In contrast, eukaryotes and many eubacteria produce glutathione (GSH). This suggests that the enzymes involved in the mycothiol biosynthetic pathway may be potential targets for selective antimicrobial chemotherapy.MSH is synthesized via a series of four unique enzyme-catalyzed reactions (6-9), as illustrated in Scheme 1. Mycothiol is reversibly oxidized by cellular oxidants and can react with electrophiles to form the S-conjugates. A detailed biochemical characterization of the cysteine ligase (MshC) from Mycobacterium smegmatis, which is the penultimate enzyme in mycothiol (15), and acyl-and aryl-CoA synthetases/ligases (16-18) catalyze reactions that generally can be divided into two halves. In the first halfreaction, one substrate reacts with ATP to form an adenylate and inorganic pyrophosphate; in the second half-reaction the second substrate reacts with the adenylated substrate...

The DinB Superfamily Includes Novel Mycothiol, Bacillithiol, and Glutathione S-Transferases

“…The Ni could be readily exchanged with Zn by dialysis and we assume the Zn form is the native form of the enzyme. In MshC Zn binds the cysteine sulfhydryl in the mycothiol ligase reaction that links Cys with GlcN-Ins producing Cys-GlcN-Ins, an intermediate in MSH biosynthesis (56, 57). The Zn ligand in YfiT (BsBST) is a possible binding site for the cysteine sulfhydryl of bacillithiol.…”

Homocysteine Editing, Thioester Chemistry, Coenzyme A, and the Origin of Coded Peptide Synthesis †

“…The first system includes mycothiol synthase MshC, a Class I CysRS paralogue that functions independently of tRNA. MshC activates Cys with ATP, forming Cys~AMP, and then transfers the activated Cys to an amino group of glucosamine in the mycothiol biosynthetic pathway [79,80]. MshC shares 36.1% primary sequence identity to CysRS.…”

“…Crystallographic structure of MshC is similar to that of CysRS and other Class I AARSs with the catalytic Rossman-fold domain of five-stranded parallel β-sheet surrounded by α-helices [81]. A superposition of MshC and CysRS structures, excluding the anticodon-binding domain of CysRS, shows an RMSD of 2.70 Å for overlapping α-carbon atoms [80]. …”

Metallobiology of Tuberculosis

“…The number of Zn containing proteins identified in mycobacteria has increased significantly as more protein structures are resolved. Zn is part of M. tuberculosis zinc-metallopeptidases (33, 34), carbonic anhydrase (35), fructose biphosphate aldolase Fba (36), the helicase RqlH (37), the cytidine deaminase Cda (38), the MshC ligase involved in mycothiol biosynthesis (39), the 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase IspF (40), the 2-isopropylmalate synthase LeuA involved in leucine biosynthesis (41), the superoxide dismutase (SOD) SodC (42, 43), the Esx-3 substrate EsxG-EsxH complex (44), the inositol 1-phosphate synthase (45), the RecA intein (46) and several more.…”