Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase

Abstract

Thiamin pyrophosphate 1 (Figure 1A) is an essential cofactor in all living systems1. Its biosynthesis involves the separate syntheses of the pyrimidine 2 and thiazole 3 precursors, which are then coupled2. Two biosynthetic routes to the thiamin thiazole have been identified. In prokaryotes, five enzymes act on three substrates to produce the thiazole via a complex oxidative condensation reaction, the mechanistic details of which are now well established2–6. In contrast, only one gene-product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae)7. Identification of three adenylated metabolites (structures 5, 12 and 17 in Figure 1B), co-purifying with THI4p, provided three molecular snapshots of the reaction pathway catalyzed by this protein. In addition, two partially active mutants were identified (C204A and H200N), which catalyzed the conversion of NAD (nicotinamide adenine dinucleotide) 6 and glycine 9 to an advanced intermediate 128. A mechanism for thiazole formation, consistent with these observations, is outlined in Figure 1B.8–11 However, the source of the thiazole sulfur remained elusive, precluding us from deciphering the subsequent steps leading to the adenylated thiazole 5. Here we report the preparation of fully active recombinant wild type THI4p, the identification of an iron-dependent sulfide transfer reaction from the protein to a reaction intermediate and the demonstration that THI4p is a suicidal enzyme undergoing only a single turnover.