Mycothiol Biosynthesis and Metabolic Functions

University Of California-San Diego, La Jolla CA

Investigators

Abstract

Mycothiol (MSH, AcCys-GlcN-Ins), is the major low-molecular-weight thiol produced by actinomycetes, a broad group of gram positive bacteria which include the streptomycetes and mycobacteria. Current evidence indicates that MSH has functions in actinomycetes analogous to glutathione (GSH) in GSH-producing organisms but our understanding of MSH biochemistry is still limited. The four enzymes involved in MSH biosynthesis and one enzyme involved in a novel MSH-dependent detoxification pathway have been identified in this laboratory. Mycobacterial mutants have also been obtained which are blocked at various steps of MSH biosynthesis. The present studies continue the elucidation of the metabolic biochemistry of mycothiol. The regulation of MSH biosynthesis will be examined through kinetic studies with the final two enzymes of MSH biosynthesis, MshC and MshD, and through gene replacement studies to test the role of putative MSH regulatory genes. The importance of MSH and its biosynthetic intermediates for mycobacterial sensitivity to toxins and antibiotics will be determined through studies of MSH deficient mutants blocked at various steps of MSH production. The role of MSH in the detoxification of the antibiotic rifamycin will be examined using radioactive labeling of MSH in Mycobacterium smegmatis to permit identification of MSH adducts and degradation products, and through kinetic studies of MSH-rifamycin adduct metabolism by the degradative enzyme mycothiol S-conjugate amidase. A novel antioxidant named mycothiol has been found to be produced by a class of bacteria which include the streptomycetes, the main antibiotic-producing bacteria, and the mycobacteria, the causative agents of tuberculosis and leprosy. Evidence indicates that mycothiol is involved in protecting cells against various toxins and antibiotics. This research will elucidate the factors involved in regulating mycothiol production in mycobacteria and the role of mycothiol in detoxifying the antibiotic rifamycin. The results could provide the basis for devising methods to enhance the ability of bacteria to detoxify environmental pollutants (bioremediation) and for increasing antibiotic production in streptomycete fermentations.