Abstract Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in my- cothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn5 trans- poson mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its pre- cursor, 1D-myo-inosityl 2-L-cysteinylamido-2-deoxy-α- D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys- GlcN-Ins acetyltransferase (mycothiol synthase, MshD) ac- tivity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuber- culosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene. Keywords mshD · Mycothiol synthase · Mycothiol · Actinomycetes · Mycobacteria Abbreviations MSH Mycothiol · MshD Mycothiol synthase · Cys-GlcN-Ins D-myo-Inosityl 2-L-cysteinylamido-2-deoxy-α-D-glucopyranoside · mBBr Monobromobimane · RSmB Bimane derivative of the thiol RSH Introduction Mycothiol (MSH) is the major low-molecular-weight thiol found in most actinomycetes, including mycobacteria and streptomycetes, but not in other bacteria or in eukaryotes (Newton et al. 1996). Its structure, 1-D-myo-inosityl-2- (N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside (AcCys-GlcN-Ins), makes it resistant to heavy-metal-cat- alyzed autoxidation (Newton et al. 1995) and it appears to have functions analogous to those of glutathione, which is not found in actinomycetes. A mycothiol-dependent formaldehyde dehydrogenase has been identified (Misset- Smits et al. 1997; Norin et al. 1997). Mycobacterium smegmatis mutants defective in MSH biosynthesis exhibit enhanced sensitivity to hydrogen peroxide and modi- fied sensitivity to antibiotics (Newton et al. 1999). Alkyl- ating agents are detoxified by mycothiol and the result- ing S-conjugates cleaved by an amidase to produce the N-acetylcysteine derivative (mercapturic acid), which is excreted from the cell (Newton et al. 2000b). A mycothiol disulfide reductase maintains mycothiol in the reduced state (Patel and Blanchard 1999, 2001). These observa- tions indicate that MSH plays a key role in protecting cells against a variety of challenges and suggest that en- zymes of MSH metabolism could be possible targets for development of new drugs and vaccines directed against Mycobacterium tuberculosis and other infectious actino- mycetes. Stopping the production of MSH should eliminate the MSH-dependent protective mechanisms, and this makes the enzymes of mycothiol biosynthesis of special interest. MSH has been shown to be produced from GlcNAc-Ins via the steps shown in Fig. 1 (Anderberg et al. 1998; Bornemann et al. 1997; Newton et al. 2000a). The details involved in the production of GlcNAc-Ins by MshA in the first step of mycothiol biosynthesis are still uncertain. A deacetylase designated MshB has been cloned and ex- pressed from M. tuberculosis H37Rv (Rv1170) and shown to convert the MshA product, GlcNAc-Ins, to the free amine, GlcN-Ins (Newton et al. 2000a). GlcN-Ins is then Teresa Koledin · Gerald L. Newton · Robert C. Fahey Identification of the mycothiol synthase gene (mshD ) encoding the acetyltransferase producing mycothiol in actinomycetes Received: 20 February 2002 / Revised: 7 May 2002 / Accepted: 11 June 2002 / Published online: 15 August 2002 ORIGINAL PAPER T. Koledin · G.L. Newton · R.C. Fahey (✉) Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093–0506 USA e-mail: rcfahey@ucsd.edu, Tel.: +1-858-5342163, Fax: +1-858-5344864 Arch Microbiol (2002) 178 : 331–337 DOI 10.1007/s00203-002-0462-y © Springer-Verlag 2002