The recent discovery of W-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"-methylhistidine to ovothiol A. (b) Ovothiol A was poorly separated from a component which was labeled by ['"Clhistidine and by ["S]cysteine, but not by [methyl-'Hlmethionine. (c) Dialysed crude extracts of C. fasciculutu catalysed the conversion of histidine to a thiolated species in the presence of pyridoxal phosphate, iron and cysteine in the absence of S-adenosylniethionine. The product of the in vitro reaction was isolated as the bimane derivative. Structural analysis using 'H and "C-NMR spectroscopy confirmed its identity as the bimane derivative of 4-mercaptohistidine.Keywords: Crithidiu fasciculatu ; ovothiol A ; 4-mercaptohistidine ; NMR.While trypanosomatids are deficient in the anti-oxidant enzymes, catalase and glutathione peroxidase [l -31, they possess abundant amounts of low-molecular-mass thiols, some of which have no counterpart in their hosts. All members of the Kinetoplastida investigated produce variable amounts of trypanothione and its immediate precursor, glutathionylspermidine [4]. The enzyme trypanothione reductase, which substitutes for glutathione reductase in these organisms, has attracted much attention as a potential drug target [5-71. Moreover, the action of a number of antitrypanosomal drugs can, at least in part, be attributed to an interaction with the trypanothione system [8, 91.More recently we have reported on the presence of ovothiol A in the trypanosomatidae [lo, 111. The ovothiols, a family of 4-mercaptohistidines, were first discovered in the egg cells and ovaries of marine invertebrates [12-141. These organisms lack glutathione peroxidase and it was, therefore, proposed that the presence of high levels of ovothiols serves to protect the egg cell against reactive oxygen intermediates released in the respiratory burst triggered by fertilization [15]. Consistent with this hypothesis the results of a number of studies indicated that ovothiols have exceptional anti-oxidant properties [16, 171.The discovery of significant amounts of ovothiol A in Crithidia fasciculata afforded an opportunity to study the metabolism and function of ovothiol A in organisms which can conveniently be cultured in large quantities and in defined growth media.The biosynthesis of ovothiol A (Scheme 1) requires methylation at N1 of the imidazole ring and introduction of a thiol group at C4. Well characterized transsulfurations, such as the interconversion of cysteine and homocysteine, proceed via pyridoxal-phosphate-catalyzed elimi...

Changes in composition of the principal low molecular mass thiols of Leishmania donovani were monitored during the transformation of promastigotes, first to stationary phase metacyclic forms and then to amastigotes. No consistent variation in the thiol composition of the parasite which could account for the known increase in resistance of metacyclic and amastigote lifecycle forms to oxidant stress could be established. Amastigotes cultivated at 37 degrees C also produced ovothiol A, as judged by incorporation of radiolabel from [3-methyl]methionine and [14C]histidine, and the incorporation of radiolabel from [35S]cysteine into ovothiol A represented about 10-15% of the total label recovered in ovothiol A, glutathione and trypanothione. Amastigotes were less susceptible than promastigotes to the effects of the redox cyclers paraquat and menadione and grew in culture in the presence of up to 20 mM buthionine sulfoximine, which completely blocked the synthesis of glutathione and its spermidine conjugates. Glutathione and trypanothione biosynthesis is, therefore, not necessary for the replication of L. donovani amastigotes in culture. Inhibition of the formation of glutathione and trypanothione did not result in an upregulation of ovothiol A production.

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