Studies on the biosynthesis of ovothiol A. Identification of 4-mercaptohistidine as an intermediate

The recent discovery of N1-methyl-4-mercaptohistidine (ovothiol A), a small aromatic thiol, in Crithidia fasciculata made it possible to study its biosynthesis in an organism which can be cultured in large quantities and under defined growth conditions. Radiolabeling experiments using intact cells indicated that the methyl group in ovothiol A is derived from methionine, while 35S was incorporated from either cysteine or methionine. Three lines of evidence suggested that transsulfuration preceded the methylation step: (a) Crithidia fasciculata failed to convert radiolabeled N pi-methylhistidine to ovothiol A. (b) Ovothiol A was poorly separated from a component which was labeled by [14C]histidine and by [35S]cysteine, but not by [methyl-3H] methionine. (c) Dialysed crude extracts of C. fasciculata catalysed the conversion of histidine to a thiolated species in the presence of pyridoxal phosphate, iron and cysteine in the absence of S-adenosylmethionine. The product of the in vitro reaction was isolated as the bimane derivative. Structural analysis using 1H and 13C-NMR spectroscopy confirmed its identity as the bimane derivative of 4-mercaptohistidine.