Abstract 2314 Phosphomethylpyrimidine synthase (ThiC): A “Radical Dance” in bacterial thiamin biosynthesis

This is the authors' abstract. We don't add key points for this paper.

Thiamin pyrophosphate (TPP, an active form of vitamin B1) is an essential cofactor in all realms of life. Grain products are the primary source of TPP and the recommended daily allowance for humans is 1.2 mg. While structurally simple, its biosynthesis is remarkably complex and presents a treasure trove of new biological chemistry. The TPP biosynthetic pathway involves separate biosynthesis of thiazole and pyrimidine moieties, which are then coupled to form TPP. The mechanistic enzymology of thiamin thiazole biosynthesis has been studied in detail. In contrast, the mechanism of thiamin pyrimidine formation is yet to be established. In bacteria, phosphomethylpyrimidine synthase (ThiC), is a radical S-adenosyl-L-methionine (SAM) enzyme that catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-amino-5-hydroxymethyl-2-methyl pyrimidine phosphate (HMP-P). This transformation is the most complex radical rearrangement in primary metabolism, with the enzyme controlling the reactivity of at least 15 radical intermediates during the transformation. Despite our extensive efforts over the last two decades, the mechanism of this chemical transformation remains elusive. Herein, we have proposed a mechanism for the ThiC-catalyzed reaction involving previously unknown reactivity of 5'-nucleotide radicals. The early steps of the rearrangement involve a mesolytic cleavage to generate a sugar-based radical, followed by radical recombination to generate a desired C-C bond. To support this mechanism, we have used site-directed mutagenesis, substrate analogs, isotopologues, derivatization strategies, and EPR to trap and characterize two early-stage intermediates in the mechanism. Site-directed mutagenesis of a conserved cysteine residue in ThiC traps a late-stage intermediate, suggesting a novel cysteine-mediated decarbonylation in this complex radical rearrangement. We have also identified the stereospecificity of initial hydrogen abstraction by 5'-deoxyadenosyl radical and identified SAM as the source of a previously unidentified hydrogen atom of thiamin pyrimidine. These intermediates provide mechanistic snapshots of the early, middle, and late stages of the ThiC-catalyzed reaction. Using bioinformatics, we have also identified ThiC-like enzymes that are involved in the biosynthesis of other natural products. This research was supported by a grant from the National Institutes of Health (DK44083) and by the Robert A. Welch Foundation (A0034).