title: The thiamine biosynthetic enzyme ThiC catalyzes multiple turnovers and is inhibited by S-adenosylmethionine (AdoMet) metabolites.
abstract: ThiC (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase; EC 4.1.99.17) is a radical S-adenosylmethionine (AdoMet) enzyme that uses a [4Fe-4S](+) cluster to reductively cleave AdoMet to methionine and a 5'-deoxyadenosyl radical that initiates catalysis. In plants and bacteria, ThiC converts the purine intermediate 5-aminoimidazole ribotide to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, an intermediate of thiamine pyrophosphate (coenzyme B1) biosynthesis. In this study, assay conditions were implemented that consistently generated 5-fold molar excess of HMP, demonstrating that ThiC undergoes multiple turnovers. ThiC activity was improved by in situ removal of product 5'-deoxyadenosine. The activity was inhibited by AdoMet metabolites S-adenosylhomocysteine, adenosine, 5'-deoxyadenosine, S-methyl-5'-thioadenosine, methionine, and homocysteine. Neither adenosine nor S-methyl-5'-thioadenosine had been shown to inhibit radical AdoMet enzymes, suggesting that ThiC is distinct from other family members. The parameters for improved ThiC activity and turnover described here will facilitate kinetic and mechanistic analyses of ThiC.
authors.affiliation: From the Department of Microbiology, University of Georgia, Athens, Georgia 30602.
authors.forename: Lauren D
authors.initials: LD
authors.lastname: Palmer
authors.affiliation: From the Department of Microbiology, University of Georgia, Athens, Georgia 30602. Electronic address: dmdowns@uga.edu.
authors.forename: Diana M
authors.initials: DM
authors.lastname: Downs
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D007700:Kinetics; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048373:Treponema denticola / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C105797:ThiC protein, Bacteria; D012436:S-Adenosylmethionine; D013835:Thiamine Pyrophosphate
keywords: Enzyme Inhibitors; Iron-Sulfur Protein; Radicals; S-adenosylmethionine (AdoMet); Thiamine; Thiamine Biosynthesis