Mechanistic Studies on the Single-Turnover Yeast Thiamin Pyrimidine Synthase: Characterization of the Inactive Enzyme

Abstract

The eukaryotic thiamin pyrimidine synthase, THI5p, has been identified as a suicidal/single-turnover enzyme that catalyzes the conversion of its active site histidine and lysine-bound pyridoxal phosphate (PLP) to the thiamin pyrimidine (HMP-P).Here we identify the histidine and PLP fragments using bottom-up proteomics and LC-MS analysis. We also identify the active form of the iron cofactor and quantitate the oxygen requirement of the THI5p reaction. This information is integrated into a mechanistic proposal for this remarkable reaction.

Oxidative Dearomatization of PLP in Thiamin Pyrimidine Biosynthesis in Candida albicans

“…A retro-Diels–Alder reaction generates the heterocycle 12 , which undergoes iterative oxidation/hydrolysis reactions ultimately to give HMP-P ( 3 ) and the observed histidine and PLP byproducts ( 4 – 6 ). This mechanistic proposal is consistent with the structure of the enzyme–substrate complex, the identification of the PLP and His-derived byproducts, with labeling studies that establish the origin of all of the HMP-P nonhydrogen atoms, and with the Fe­(II) and oxygen requirements of the reaction. ,,− …”

“…THI5p is a challenging experimental system involving unprecedented PLP and histidine reactions. The turnover is low (∼0.3) because most of the enzyme (>60%) has undergone inactivation resulting from HMP-P ( 3 ) biosynthesis during overexpression . Therefore, a nontraditional strategy was required to elucidate its mechanism.…”

“…The reaction requires Fe­(II) and oxygen (Figure ). , A mechanistic hypothesis for this complex reaction is shown in Figure . In this mechanism, PLP has been activated for a Diels–Alder reaction − with the imidazole of His66 by an oxidative dearomatization to azadiene 10 .…”

“…The microbial biosynthesis of thiamin pyrophosphate involves the coupling of the thiazole and pyrimidine heterocycles. − In yeast, the thiazole synthase (THI4p) and the pyrimidine synthase (THI5p) both use enzyme-derived atoms for product formation and are single-turnover enzymes. − While the mechanism of the thiazole synthase is relatively well-understood, mechanistic understanding of the pyrimidine synthase is still at an early stage. ,− This enzyme uses the active site His66 ( 1 ) and the PLP imine ( 2 ) with Lys62 to form the thiamin pyrimidine (HMP-P, 3 ). The reaction requires Fe­(II) and oxygen (Figure ).…”

“… 6 , 8 A mechanistic hypothesis for this complex reaction is shown in Figure 2 . 8 In this mechanism, PLP has been activated for a Diels–Alder reaction 14 − 16 with the imidazole of His66 by an oxidative dearomatization to azadiene 10 . A retro-Diels–Alder reaction generates the heterocycle 12 , which undergoes iterative oxidation/hydrolysis reactions ultimately to give HMP-P ( 3 ) and the observed histidine and PLP byproducts ( 4 – 6 ).…”