| dc.description.abstract | Thiamin pyrophosphate is an essential cofactor in all living systems. Its biosynthesis involves two separate pathways to synthesize pyrimidine and thiazole, followed by a coupling of these two heterocycles to generate thiamin. In this dissertation, the Bacillus subtilis thiazole synthase (ThiG) and Candida albicans pyrimidine synthase (THI5p) were investigated.
The unanticipated reverse reaction of ThiG and the thiazole tautomer generated an intermediate, which was characterized by mass spectrometry and further applied to investigate the later steps of thiazole biosynthesis involving ThiG and ThiO, which is the glycine oxidase. This study demonstrated our proposed mechanism of B. subtilis thiazole biosynthesis involving ThiG and ThiO.
The THI5 protein (THI5p) was reconstituted in vitro and identified as a single turnover protein to use an active-site histidine to react with pyridoxal 5’-phosphate (PLP) to generate HMP-P. The histidine structure after HMP-P formation was characterized as a keto-acid. The PLP by-products were trapped by phenylhydrazine derivatization. The structural characterization identified as glyoxylate via C3-fragment. The keto-acid, C3-fragment and glyoxylate had oxygen incorporations from O2. The pattern of incorporations suggested that the protein utilized multiple O2 to synthesize HMP-P. Moreover, a shunt product, PLP-nitrile, was identified to suggest that the reaction was initiated by a radical reaction. Based on these preliminary information, a mechanism was proposed. To investigate the proposed mechanism, phosphopeptide enrichment was used to identify the peptides containing possible shunt intermediates. The investigations could help to test the proposed mechanism.
Because THI5p required Fe(II) and O2 to catalyze HMP-P synthesis, preliminary bioinorganic investigations were conducted by oxygen consumption measurements, EPR, and UV-Vis spectra. The results suggested that the protein used multiple O2 and might be a diiron protein. All of the information could serve as a stepping stone for future studies.
All of the investigations could help us to understand how nature designs THI5p to be a single turnover protein to synthesize HMP-P without chemical and biochemical precedent. | en |
