Nature 478, 542–546 (2011)

Thiamine pyrophosphate (TPP), an enzymatic cofactor, is assembled by a convergent strategy that joins a core thiazole scaffold with a pyrimidine side chain. The biosynthetic route to the thiazole component in prokaryotes is well understood, but, curiously, in eukaryotes only one enzyme (known in Saccharomyces cerevisiae as THI4p) is responsible for the multistep conversion of nicotinamide adenine dinucleotide (NAD) and glycine to the thiazole scaffold. Chatterjee et al. now demonstrate that the sulfur atom of TPP is derived from THI4p itself. Biochemical analysis showed that THI4p copurifies with three thiazole biosynthetic intermediates and that its activity is enhanced in the presence of Fe(II). MS characterization revealed that the molecular mass of expressed THI4p was 34 Da lower than predicted; reanalysis of earlier THI4p crystallographic data revealed an absence of sulfur electron density at Cys205 and evidence for a dehydroalanine residue at this position. Taken together, these observations suggested that during catalysis THI4p covalently captures biosynthetic intermediates at Cys205, and the linking sulfur atom is transferred into the substrate through β-elimination. The successful reconstitution of thiazole biosynthesis in yeast provides compelling evidence that THI4p is a stoichiometric cosubstrate that, in addition to its other roles, acts as a sacrificial sulfur donor in TPP biosynthesis.