Thiamin is an essential component of the human diet with an RDA of 1.2 mg. It is also an important commercial chemical and it is widely used as a food additive and flavoring agent. Annual production, by chemical synthesis, is on the order of 3,300 tons. Thiamin-dependent enzymes play an important role in carbohydrate and branched-chain amino acid metabolism. At this time, our mechanistic understanding of this class of enzymes is sophisticated. In contrast, while thiamin was the first vitamin identified, our understanding of its biosynthesis is still incomplete because the biosynthetic pathway is complex and involves unprecedented reaction chemistry. Thiamin consists of a thiazole linked to a pyrimidine. The thiazole moiety in bacteria is formed from deoxy-D-xylulose-5-phosphate, glycine, and a 66 amino acid protein thiocarboxylate via a complex oxidative condensation. The pyrimidine in bacteria is formed from 5-aminoimidazole ribonucleotide via a complex rearrangement reaction. The pyrimidine is then coupled to the thiazole to give thiamin phosphate and a final phosphorylation gives the biologically active form of vitamin B1. Our mechanistic understanding of thiazole formation in bacteria is now at an advanced stage and we have recently established some of the main features of thiazole formation in eukaryotes. The pyrimidine thiazole coupling reaction is also well understood and the pyrimidine carbocation intermediate has been structurally and kinetically characterized. In contrast our mechanistic understanding of the remarkable chemistry involved in the formation of the thiamin pyrimidine in bacteria and in eukaryotes is still at an early stage. In the next funding period, we propose to continue our mechanistic characterization of the thiazole biosynthetic enzymes, evaluate the generality of the novel sulfur transfer chemistry involved in thiazole formation, and carry out mechanistic and structural studies on the pyrimidine biosynthetic enzymes. In addition, we will explore the enzymology of the salvage of acid-degraded thiamin. The long-term goal of our research is the complete mechanistic understanding of the enzymology of thiamin biosynthesis in both prokaryotes and eukaryotes and the elucidation of connections between thiamin metabolism and other aspects of cellular physiology. Our studies are significant for four reasons. First, it is important to understand how thiamin is biosynthesized because this vitamin is a required component of the human diet and an essential cofactor for all forms of life. Second, the biosynthetic pathway involves an unusually large amount of unprecedented biological chemistry. Third, our studies will facilitate the construction of overexpression strains that will be of use for the commercial production of thiamin by fermentation. Finally, inhibitors of thiamin biosynthetic enzymes may provide a selective strategy for antibiotic design in bacteria such as Mycobacterium tuberculosis that lack a thiamin transport system.

Public Health Relevance

We are interested in how vitamin B1 (thiamin) is assembled in living systems. This is an important problem because vitamin B1 is a required component of the human diet and is essential for all forms of life. Thiamin is added to many foods and our studies will facilitate its commercial production by fermentation. In addition, our research has potential applications in the design of TB specific antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK044083-22
Application #
8289416
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Sechi, Salvatore
Project Start
1991-09-15
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
22
Fiscal Year
2012
Total Cost
$337,425
Indirect Cost
$107,101
Name
Texas A&M University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
020271826
City
College Station
State
TX
Country
United States
Zip Code
77845
Zhang, Xuan; Eser, Bekir E; Chanani, Prem K et al. (2016) Structural Basis for Iron-Mediated Sulfur Transfer in Archael and Yeast Thiazole Synthases. Biochemistry 55:1826-38
Xu, Hui; Chakrabarty, Yindrila; Philmus, Benjamin et al. (2016) Identification of the First Riboflavin Catabolic Gene Cluster Isolated from Microbacterium maritypicum G10. J Biol Chem 291:23506-23515
Eser, Bekir E; Zhang, Xuan; Chanani, Prem K et al. (2016) From Suicide Enzyme to Catalyst: The Iron-Dependent Sulfide Transfer in Methanococcus jannaschii Thiamin Thiazole Biosynthesis. J Am Chem Soc 138:3639-42
Nemeria, Natalia S; Shome, Brateen; DeColli, Alicia A et al. (2016) Competence of Thiamin Diphosphate-Dependent Enzymes with 2'-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin. Biochemistry 55:1135-48
Mehta, Angad P; Abdelwahed, Sameh H; Fenwick, Michael K et al. (2015) Anaerobic 5-Hydroxybenzimidazole Formation from Aminoimidazole Ribotide: An Unanticipated Intersection of Thiamin and Vitamin B₁₂ Biosynthesis. J Am Chem Soc 137:10444-7
Mehta, Angad P; Abdelwahed, Sameh H; Mahanta, Nilkamal et al. (2015) Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions. J Biol Chem 290:3980-6
Fenwick, Michael K; Mehta, Angad P; Zhang, Yang et al. (2015) Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase. Nat Commun 6:6480
Mehta, Angad P; Abdelwahed, Sameh H; Begley, Tadhg P (2015) Molybdopterin biosynthesis-Mechanistic studies on a novel MoaA catalyzed insertion of a purine carbon into the ribose of GTP. Biochim Biophys Acta 1854:1073-7
Philmus, Benjamin; Decamps, Laure; Berteau, Olivier et al. (2015) Biosynthetic versatility and coordinated action of 5'-deoxyadenosyl radicals in deazaflavin biosynthesis. J Am Chem Soc 137:5406-13
Cooper, Lisa E; O'Leary, Seán E; Begley, Tadhg P (2014) Biosynthesis of a thiamin antivitamin in Clostridium botulinum. Biochemistry 53:2215-7

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