Characterization and total synthesis of mycothiol: Exploring roles of thiols with Nocardia sp. nitric oxide synthase

Mycothiol (MSH) is the major low molecular weight thiol found in most actinomycetes including mycobacteria and streptomycetes. MSH has functional similarities with glutathione, the most common eukaryotic thiol.; Bacterial nitric oxide synthase (NOSNOC) capable of generating nitric oxide (NO) was purified and characterized from Nocardia sp. NRRL 5646 in our laboratory. Thiol-containing compounds react with NO producing S-nitrosothiols, stable and storage forms of NO. The nature and types of thiols possibly related to NO in Nocardia are unknown.; To investigate roles of thiols with NO in Nocardia sp., we quantified thiols and S-nitrosothiols and demonstrated that MSH is the major low molecular weight thiol in this organism. MSH was isolated as its fluorescent bimane derivative, the first purification and characterization of MSH-bimane from Nocardia sp. We observed a decrease in MSH concentrations in cell extracts when Nocardia was grown in media containing NOS substrate and cofactor. Increasing NOSNOC activity by adding L-arginine and tetrahydrobiopterin influenced intra- and extracellular NO2- and NO 3- concentrations. Results suggest that MSH may play a role in NO regulation and detoxification in Nocardia.; Glutathione-dependent formaldehyde dehydrogenase was involved in the metabolism of S-nitrosoglutathione and was assigned as S-nitrosoglutathione reductase in E. coli, S. cerevisiae and mouse macrophages. Recently, MSH-dependent formaldehyde dehydrogenase in M. tuberculosis was also identified as a S-nitrosomycothiol reductase. We found that Nocardia cell extracts contain MSH-dependent formaldehyde dehydrogenase with a specific activity 0.015 units/mg protein. However, S-nitrosomycothiol reductase activity could not be detected using artificial substrates, glyco-SNAP2 which has structural similarities with S-nitrosomycothiol, or S-nitrosoglutathione as substrates.; Free MSH has never been available either from natural or synthetic sources. We achieved the first total synthesis of MSH and MSH-disulfide to study the possible relationship of MSH to NO in Nocardia. We report full spectral characterization for underivatized MSH and confirmed its structure by making the known MSH-bimane derivative.