This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Yeast: Thi4 produces the thiazole moiety in thiamin in yeast. An ortholog of Thi4 in Neurospora crassa binds to the peptidyl-prolyl cis/trans isomerase NcCyP41. NcCyP41 is believed to be responsible for thiamin biosynthesis by inducing a conformational change in Thi4 for product release. The protein Thi5 is required for the biosynthesis of the pyrimidine moiety of thiamin. Thi5 uses pyridoxine and histidine as substrates. Bacteria: TenA has been recently identified as a thiaminase II, which degrades thiamin to pyrimidine and thiazole. The structure of TenA from B. subtilis was determined using data collected at NE-CAT 8BM and the paper was published in Biochemistry in 2005 (A.V. Toms). Key active site residues were mutated based on the structure. Our goal is to determine the structure of the mutants complexed with the substrate thiamin to elucidate the mechanism. The structure complex of ThiSF was solved, and subsequently published in Biochemistry (Lehmann). ThiL, thiamine monophosphate kinase, is the last enzyme in the thiamine biosynthetic pathway. It is responsible for the phosphorylation of thiamine phosphate to yield thiamine pyrophosphate. The goal of this project is to determine the binding mode of the ATP responsible for phosphorylation using the non-hydrolyzable analog AMP-PCP as well as how thiamin monophosphate and thiamin pyrophosphate bind to ThiL for catalysis. ThiC catalyzes the conversion of AIR (5-aminoimidazole ribotide) to the pyrimidine moiety of thiamin, which is the most complex unsolved mechanistic problem in primary metabolism. ThiI and ThiS are proteins involved in the synthesis of the thiazole moiety of thiamin. ThiI may play a role in the sulfur transfer to adenylated ThiS. ThiK, thiamin kinase, is responsible for the salvage of thiamin from the growth medium. DXP (1-deoxy-D-xylulose-5-phosphate) synthase catalyzes the formation of DXP, the precursor to the thiazole moiety of thiamin, from pyruvate and glyceraldehyde-3-phosphate.
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