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

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 biosynthetic pathway, has been performed (10, 11). MshC catalyzes the ATP-dependent condensation of cysteine and GlcN-Ins via a Ping Pong kinetic mechanism (Scheme 2). It has been proposed based on sequence comparisons that MshC is related to cysteinyl-tRNA synthetase (CysRS), and shares a common evolutionary origin (12). Adenylate-forming enzymes, including class I and II aminoacyl-tRNA synthetases (AaRS) (13), pantothenate synthetase (14), malonyl-CoA synthetase (15), and acyl- and aryl-CoA synthetases/ligases (16-18) catalyze reactions that generally can be divided into two halves. In the first half-reaction, 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 to generate the product and AMP. The three-dimensional structures of adenylate-forming enzymes comprise multiple domains that are usually linked by flexible loops to allow conformational changes upon substrate binding or domain alternation. In class I CysRS's, binding of cysteine induces a significant conformational change including the movement of the zinc ion towards the substrate, the movement of the conserved W205 indole ring to interact with the cysteine side chain, as well as the closure of the loop which contains the KMSKS signature motif (19). In the case of 4-chlorobenzoate:CoA ligase, the C-terminal domain rotates by ∼140° to permit alternate catalysis of adenylation and thioester formation reactions (20). Such a large conformational change has been termed “domain alternation”, and was first introduced to describe such structural changes in B12-dependent methionine synthetase (21). Scheme 1 Mycothiol biosynthesis. The MshA-D enzymes catalyze a glycosyltransferase, deacetylase, a ligase and an acetytransferase reaction, respectively. The phosphatase is depicted as being unknown at present. Scheme 2 The reactions catalyzed by MshC. MshC from M. smegmatis has been cloned, expressed, purified and kinetically characterized (11). The enzyme is a monomer with a molecular weight of ∼ 47 kDa. A stable analog of the intermediate cysteineadenylate, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine (CSA), exhibits competitive inhibition versus ATP, with an inhibition constant of ∼300 nM versus ATP. Previous crystallization efforts of MshC, using a variety of screening conditions and with a number of different molecular constructs failed to produce protein crystals. Limited proteolysis has been documented as a powerful tool to obtain structural information on stable domains in proteins that have resisted crystallization (22, 23). It has been observed that limited proteolysis occurs exclusively as solvent-accessible and flexible regions of exemplary proteins (24). These flexible regions include disordered N- and C-termini as well as exposed loops or linkers between domains (25), which could be the cause for unsuccessful crystallization. Limited proteolysis generally yields a nicked or cleaved species with the overall fold of the native protein remaining intact. The products of proteolysis often exhibit higher solubility and are often amenable to high-resolution structural analysis (22, 23). We report here the limited proteolysis of the MshC-CSA complex that yields a single cleaved product that readily crystallized, and the three-dimensional structure of the MshC-CSA complex at 1.6 A resolution.