A novel antioxidant, mycothiol, has recently been shown to be produced by most actinomycetes, a class of bacteria encompasing a major fraction of soil bacteria. These include disease-producing mycobacteria, such as Mycobacterium tuberculosis, and the streptomycetes, a major source of antibiotics. Mycothiol has been shown to be unusually resistant to autooxidation and chemical mutants of M. smegmatis blocked in mycothiol biosynthesis have been found to exhibit enhanced sensitivity to hydrogen peroxide. Whether these enhanced sensitivities result solely from blockage of mycothiol biosynthesis or from other mutations which might also be present is uncertain. The present studies will test the hypothesis that mycothiol, although not essential for laboratory survival of mycobacteria, plays a key role in protecting actinomycetes against oxidative and antibiotic challenge. Mycothiol biosynthesis involves the assembly of a pseudodisaccharide (GlcN-Ins) by linking of glucosamine (GlcN) with myo- inositol (Ins). Cysteine (Cys) is then ligated to GlcN-Ins in an ATP- dependent reaction to produce Cys-GlcN-Ins. The latter is acetylated by a acetyl CoA-dependent transacetylase to produce mycothiol (AcCys-GlcN-Ins). This research will investigate the ligase and transacetylase steps of mycothiol biosynthesis. The enzymes will be purified and characterized with respect to their catalytic functions. The genes coding the enzymes catalyzing these reactions will be identified and mutants blocked specifically in these enzymes will be produced in the model organism M. smegmatis . These mutants will be studied to ascertain the effects of blocking mycothiol biosynthesis upon the ability of the organism to resist oxidative and antibiotic stress. These studies will provide fundamental biochemical information about two key steps in the biosynthesis of mycothiol. They will also test the role of mycothiol in the protection of mycobacteria against damage by hydrogen peroxide and other oxidants, and will establish the extent to which mycothiol can protect against antibiotics. The results could prove important in the design of new drugs directed against mycobacteria and will provide insight concerning the natural function of mycothiol in actinomycetes.
