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Thiamin Biosynthesis in Yeast – Mechanistic Studies on THI5, A Remarkable Suicide Enzyme
Abstract
Thiamin pyrophosphate is the active form of vitamin B1 or thiamin. It is an essential co-factor for all living systems and participates in biochemical reactions like carbohydrate and branched-chain amino acid metabolism. The biosynthesis of thiamin pyrophosphate is achieved differently in eukaryotes and prokaryotes, both of which involve surprisingly complex chemistry. Here in this work, we report the detailed mechanistic studies of yeast thiamin pyrimidine synthase (THI5p) that catalyzes the formation of thiamin pyrimidine (HMP-P) in a remarkable reaction. THI5p is a suicidal or single-turnover enzyme that uses its active site His66 and Lys62 bound pyridoxal phosphate (PLP) in the presence of Fe(II) and oxygen to form HMP-P. We have previously characterized the single-turnover inactivated form of the enzyme and the PLP-by products and based on that we outlined a mechanistic proposal for THI5p catalyzed reaction. Following the mechanism-guided approach and utilizing a combination of biochemical, chemical-model, and proteomics studies, we have discovered the critical intermediate in THI5p catalyzed reaction in the form of an oxidatively dearomatized PLP species (ox-PLP).
We have demonstrated the formation and reaction of this ox-PLP species in THI5p reaction by trapping it in the form of the ox-PLP-imidazole adduct in a chemical rescue experiment with THI5p-H66G. We have also established that the formation of this ox-PLP species is Fe(II) and oxygen-dependent. We provided support for the structure and formation of this ox-PLP-imidazole adduct by preparing a synthetic ox-PLP cycloaddition dimer in solution.
We have identified and characterized three new shunt products in the THI5p reaction that are proposed to be formed from an ox-PLP species generated during the THI5p reaction. The formation of these shunt products further validated our mechanistic proposal consistent with the Fe(II) and oxygen-mediated oxidative dearomatization of active site PLP. In addition, we have quantitated the oxygen consumption of THI5p reaction, determined the active form of iron co-factor, and gained mechanistic insight into the formation of PLP-by products. These studies added more clarity to the overall mechanistic proposal for this complex reaction.
Based on these key findings, we have outlined a set of future studies that will be insightful to fully comprehend the THI5p mechanism and complete our understanding of the biosynthesis of this vital co-factor.
Citation
Mondal, Anushree (2022). Thiamin Biosynthesis in Yeast – Mechanistic Studies on THI5, A Remarkable Suicide Enzyme. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /197938.