Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases

Abstract

Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.

Figure 1.

Structural features of MshD. (A) Reaction catalyzed by MshD, the final step in the biosynthesis of mycothiol. (B) Stereo Cα diagram of MshD with ribbon diagram of the MshD binary acetyl–CoA complex. In the Cα diagram every 10th residue is marked by a black sphere, while every 20th residue is numbered. In the ribbon diagram, the conserved structural elements of the GNAT fold are colored according to Vetting et al. (2002). Dashed lines indicate structural elements with no electron density. Acetyl–CoA is modeled in a stick representation. Figures 1B and 2 ▶ ▶ were prepared with the programs PYMOL (DeLano 2002) and MOLSCRIPT (Kraulis 1991).

Figure 2.

Interaction of MshD with substrates. (A) Stereodiagram of the acetyl–CoA (thick sticks) binding site in the C-terminal domain. (B) Solvent-accessible surface representation of MshD with experimentally determined acetyl–CoA (yellow carbons) and modeled Cys–GlcN–Ins (blue carbons). For visual clarity, four loops (residues 70–75, 176–179, 266–277, 226–230) that exhibit high mobility and overlay the sides of the canyon were removed prior to calculation of the surface.