Insights into the initial steps of the thiamin pyrimidine synthase (ThiC)-catalyzed reaction through EPR spectroscopic characterization of radical intermediates.

Thiamin pyrimidine synthase (ThiC) is a noncanonical radical SAM enzyme that catalyzes the complex radical rearrangement of aminoimidazole ribonucleotide (AIR) to hydroxymethylpyrimidine phosphate (HMP-P) as part of the thiamin biosynthetic pathway in bacteria and plants. In this work, we investigate the mechanism of ThiC using advanced electron paramagnetic resonance (EPR) techniques. Freeze-quenching a reaction of ThiC revealed the accumulation of a new radical species. By employing electron nuclear double resonance (ENDOR) spectroscopy with various substrate isotopologues, we determined the hyperfine parameters of several nuclei, allowing us to propose a structure for this intermediate. The accumulated species was characterized as a dihydro-aminoimidazole centered radical attached to two ribose derived fragments. This radical is sensitive to perturbations in the enzyme H-bonding network. In addition, mutagenesis of active site residues results in the accumulation of two distinct intermediates, including a C5' ribonucleotide centered radical and a ribose C2' radical fragment. Identification of these early radical intermediates provides insights into the initial steps of the ThiC mechanism. The ThiC-catalyzed reaction involves a 20-step radical cascade and is the most complex rearrangement found in biosynthesis. This study highlights the pivotal role that EPR can play in elucidating the mechanism of highly complicated enzyme-catalyzed reactions.