In Saccharomyces cerevisiae, thiamin pyrimidine (1 ) is formed from histidine and pyridoxal-5'-phosphate (PLP) (2 ). The origin of all of the pyrimidine atoms had been previously determined using labeling studies and suggested that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent.Here I report the heterologous overexpression of the closely related Candida albicans pyrimidine synthase (THI5p), and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 (3 ) in close proximity to the PLP. My data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme. The mass analysis of the peptides showed that the imidazole ring of His66 is converted to the keto-acid (4 ). Furthermore, the authentic peptide was synthesized by solid phase peptide synthesis to confirm the keto-acid structure. 18O2 studies demonstrated two oxygen atoms from different O2 molecules were incorporated into the carboxylic acid of the keto-acid. Furthermore, there is a rare protein modification that occurs during the THI5p activity assays in the absence of PLP. Hydroxylation on His66 forming 2-oxo-Histidine (5 ) was identified and O2 was incorporated into this modification. The shunt product, PLP-nitrile (6 ), was characterized which implies that pyrimidine formation involves the radical reaction. Recently, the byproduct of 2  was identified as 7  and 8  using phenhylhydrazine derivatization followed by LC-MS. The positions of oxygen incorporation were established by using isotope labeling (18O2 and 18O-water) and LC-MS/MS. In summary, thiamin pyrimidine biosynthesis involves a radical reaction and consequential oxygen activation. In the future, incorporation of unnatural amino acids at position 66 will be utilized to trap the reaction intermediates to unveil this unprecedented enzyme mechanism. 

Mechanistic studies of the mysterious pyrimidine synthase in eukaryotic thiamin biosynthesis

247th National Meeting (2014)

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