The biosynthesis of ovothiol A (N¹‐methyl‐4‐mercaptohistidine)

Abstract

Crude extracts of Crithidia fasciculata catalyse the formation of 4‐mercapto‐l‐histidine, an intermediate in the biosynthesis of ovothiol A (N1‐methyl‐4‐mercaptohistidine), in the presence of histidine, cysteine, Fe2+ and pyridoxal phosphate. This activity was present in a 35–55% ammonium sulfate fraction that was shown to produce a transsulfuration intermediate in the absence of pyridoxal phosphate. The transsulfuration intermediate was isolated and identified as S‐(4′‐l‐histidyl)‐l‐cysteine sulfoxide. The synthase activity, partially purified by anion‐exchange chromatography, was shown to require oxygen and could be used to synthesize a number of isotopically labeled S‐(4′‐l‐histidyl)‐l‐cysteine sulfoxides. Sulfoxide lyase activity was partially resolved from the synthase by anion‐exchange chromatography. The phenylhydrazone of the product derived from the cysteine moiety of the sulfoxide coeluted with the phenylhydrazone of pyruvate on HPLC, but this assignment could not be confirmed by mass spectral analysis. S‐(4′‐[14C]l‐histidyl)‐[U‐13C3,15N]l‐cysteine sulfoxide was synthesized and converted to products of the lyase reaction in the presence of lactate dehydrogenase and NADH. The 13C‐labeled product was identified by 13C‐NMR spectroscopy as lactate and the primary product of the lyase reaction is therefore pyruvate. With S‐(4′[3H]l‐histidyl)‐[14C]l‐cysteine sulfoxide as the substrate [14C]lactate, [14C]cysteine and [3H]4‐mercaptohistidine could be detected as products of the lyase reaction, but the sum of the two thiol species exceeded the amount of sulfoxide substrate used. Evidence is presented that this anomaly was due to the utilization of sulfur from dithiothreitol for the formation of cysteine.

On ovothiol biosynthesis and biological roles: from life in the ocean to therapeutic potential

“…38 This study also showed that this Tyr residue The second reaction in ovothiol biosynthesis is catalyzed by a PLP-dependent β-lyase. 39 Comparison of OvoA encoding genomes from many organisms did not reveal any gene coding for a β-lyase exclusively dedicated to ovothiol biosynthesis. Given that β-elimination of cysteine S-conjugates is a fairly simple and a very common reaction, it is possible that most organisms use unspecific β-lyases to support ovothiol production.…”

Thiohistidine Biosynthesis

“…An analogous study with cell-free extracts from Crithidia fasciculata (a kinetoplastid), suggested that ovothiol A biosynthesis proceeds in a similar manner via a 5-histidinyl cysteine sulfoxide intermediate (10, Scheme 1). [30] When we started our own research in this field, none of the proposed thiohistidine biosynthetic enzymes were known.…”

“…[25,26] Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the human pathogens which cause tropical diseases such as sleeping disease, Chagas disease and leishmaniasis also produce ovothiol A. [27][28][29][30] Elucidation of the corresponding biosynthetic pathway may reveal novel strategies to treat these conditions, for which no efficient therapy is known. Furthermore, identification of the ovothiol A biosynthetic genes (see below) revealed that several plant pathogens such as Erwinia amylophora, the causative agent of fire blight and Phytophthora infestans which causes potato blight, are also ovothiol A producers.…”

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

“…In particular, in both enzymes an iron is ligated to two histidyl imidazoles and a carboxylate side-chain. Because of their common chemistry and similar structural features, it has been suggested that they share similar catalytic mechanisms [51]. …”