The Substrate-induced Conformational Change of Mycobacterium tuberculosis Mycothiol Synthase

Abstract

The structure of the ternary complex of mycothiol synthase from Mycobacterium tuberculosis with bound desacetylmycothiol and CoA was determined to 1.8 Å resolution. The structure of the acetylCoA-binary complex had shown an active site groove that was several times larger than its substrate. The structure of the ternary complex reveals that mycothiol synthase undergoes a large conformational change in which the two acetyltransferase domains are brought together through shared interactions with the functional groups of desacetylmycothiol, thereby decreasing the size of this large central groove. A comparison of the binary and ternary structures illustrates many of the features that promote catalysis. Desacetylmycothiol is positioned with its primary amine in close proximity and in the proper orientation for direct nucleophilic attack on the si-face of the acetyl group of acetyl-CoA. Glu-234 and Tyr-294 are positioned to act as a general base and general acid to promote acetyl transfer. In addition, this structure provides further evidence that the N-terminal acetyltransferase domain no longer has enzymatic activity and is vestigial in nature.Members of the bacterial prokaryotic group termed actinomycetes lack the low molecular weight thiol, glutathione, typically found in many bacteria and in eukaryotes and instead produce mycothiol (acetyl-Lcysteine-1-D-myo-inosityl-2-deoxy-D-glucopyranoside) (1, 2). Similar to glutathione, mycothiol provides the cell protection from oxidative damage and electrophilic toxins (3, 4) and is maintained in the reduced state by a reductase (5). Mycothiol is less prone to metal ion-catalyzed autoxidation than is glutathione and may serve as a stable intracellular store of cysteine (1). Mycobacterium smegmatis mutants that were deficient in mycothiol production were hypersensitive to alkylating agents, free radicals, and antibiotics (4, 6). Mycobacteria produce an especially large amount of mycothiol, with the intercellular concentration in Mycobacterium tuberculosis reaching millimolar concentrations (7).The importance of mycothiol to mycobacterial organisms and the absence of mycothiol in eukaryotes makes the mycothiol biosynthesis pathway an attractive drug target (8). Mycothiol biosynthesis in M. tuberculosis is a multistep process involving four enzymatic reactions catalyzed by MshA, MshB, MshC, and MshD (Scheme 1). Mycothiol synthase (MshD), 2 the last enzyme in mycothiol biosynthesis, catalyzes the acetylation of the cysteinyl amine of 1-D-myo-inosityl-2-L-cysteinylamido-2-deoxy-␣-D-glucopyranoside (desacetylmycothiol (DAM)) (9). The close proximity of the relatively reactive primary amine to the active thiol would potentially yield side reactions during mycothiol reduction and conjugation reactions, thus preventing mycothiol regeneration by mycothione reductase or mycothiol S-conjugate amidase. The protection of the primary amine by acetylation is therefore presumed to be an important step in the formation of a useful detoxification agent.The previously reported structure of th...

A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

“…The GNAT domain functions as the binding site for the substrate acetyl-CoA. MshD contains two GNAT domains at amino acids 1 to 140 and at amino acids 151 to 315 (27,28). The C-terminal GNAT domain contains the catalytic site, while the N-terminal domain contributes to the structural stability of the protein (28).…”

“…Shortly thereafter, the crystal structure of the M. tuberculosis acetyl-CoA-MshD complex was reported (27), and the recent structural analysis of a ternary complex produced from CysGlcN-Ins and CoA provides insights into the conformational changes involved in catalysis (28). Transposon site hybridization studies identified this gene (Rv0819) as nonessential for the growth of M. tuberculosis (23).…”

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

“…It appears to have two separate regions with similarity to the pfam00583 domain for acetyltransferase and is approximately twice the size of homologous proteins, indicating that MshD is the result of a gene duplication (59). Blanchard and coworkers (147) Kinetic studies indicated a ternary complex mechanism common to the GNAT superfamily of N-acetyltransferases in which both substrates must be present for the reaction to occur (148). Based upon the crystal structure of the mixed disulfide of Cys-GlcN-Ins and CoA and the pH dependence of enzyme activity, the catalytic mechanism shown in Fig.…”

“…MshD is also likely to be a drugable target considering the substantial surfaces involved in the binding of the substrate moieties (148). The mshD mutant produces a small amount of MSH as well as a substantial level of formyl-Cys-GlcN-Ins, and the mshD gene is not essential for growth of M. tuberculosis (13).…”

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

“…Therefore MshA and MshC are important potential drug targets for treatment of tuberculosis and other Actinomycetales infections. The three-dimensional structures of both MshB (19) and MshD (20,21) have been determined; however, there have been no reports of structures for MshA or MshC.…”