Thiamin is essential to forms of life. Thiamin pyrophosphate plays a critical role in the combustion of carbohydrates and in the synthesis of branched-chain amino acids. Thiamin deficiency causes fatigue, confusion, depression, and irritability, and if left untreated, can be fatal. Thiamin is biosynthesized by most bacteria, yeas, fungi and plants. Humans must obtain thiamin from their diets. The overall goal of our research is to understand the detailed mechanistic enzymology of thiamin biosynthesis and metabolism. The experiments are designed to fill important gaps in our understanding of the thiamin pyrimidine synthase in bacteria and plants (ThiC), the thiamin thiazole synthase in yeast (THI4), the thiamin pyrimidine synthase in yeast (THI5), and thiamin degrading enzymes (thiaminases). ThiC is a novel radical SAM enzyme and catalyzes the complex rearrangement of aminoimidazole ribonucleotide to the thiamin pyrimidine. We have recently determined a structure of ThiC with its [4Fe-4S] cluster. Unlike canonical radical SAM enzymes the cluster binding domain is tethered to the catalytic domain, and inserts through domain swapping into the active site of a twofold related monomer. Curiously, in our initial structure the cluster is located 25 from the active site, suggesting that translocation of the domain must take place prior to catalysis. Yeast THI4 catalyzes the condensation of NAD, glycine, and cysteine to form adenylated carboxythiazole. We have shown the THI4 is an iron-dependent suicide enzyme and that the source of the thiazole sulfur atom is a cysteine side chain from THI4 itself. The structur of THI4 identified key active site residues, but we have not yet identified the iron binding site. n archaea, the yeast THI4 ortholog was shown to catalyze the isomerization is ribose 1,5-bisphosphate to ribulose 1,5-bisphosphate. We have shown that the archaeal THI4 ortholog also catalyzes synthesis of the thiamin thiazole, but uses sulfide as the sulfur source. We do not yet know if this dual activity is present in other THI4's. Yeast THI5 catalyzes a remarkable condensation of PLP and histidine to form the thiamin pyrimidine. We have shown that THI5 is an iron-dependent suicide enzyme and that the histidine side chain comes from THI5 itself. The structure of THI5 identified key active site residues, but we have not yet identified the iron binding site. For ThiC, THI4 and THI5, we will determine structures with substrates, products and analogs, identify metal binding sites, and use mutant enzymes to trap intermediates, thus filling in key mechanistic details. Thiaminase-I and II's are thiamin degrading enzymes that paradoxically often cluster with thiamin biosynthetic enzymes. The Bacillus subtilis thiaminase-II was shown to participate in thiamin salvage. We therefore hypothesize that salvage of degraded thiamin may be the general role of thiaminase-II's and that diverse thiaminases salvage different forms of degraded thiamin. We hypothesize that thiamine-I may initiate thiamin cleavage in a recently discovered thiamin catabolic pathway.

Public Health Relevance

Thiamin is essential to all forms of life. While bacteria, plants, yeast and fungi can biosynthesize thiamin, humans must obtain thiamin from their diets. Our goal is to achieve a detailed understanding of the mechanistic enzymology of thiamin biosynthesis in both prokaryotes and eukaryotes. Understanding thiamin biosynthesis may be useful for the production of thiamin by fermentation, or for the development of antibiotics that interfere with thiamin biosynthesis.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01DK067081-10
Application #
8759058
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Maruvada, Padma
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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