Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 42, Pages 30693-30699Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.500280
Keywords
Enzyme Inhibitors; Iron-Sulfur Protein; Radicals; S-adenosylmethionine (AdoMet); Thiamine; Thiamine Biosynthesis
Categories
Funding
- National Institutes of Health [GM47296]
- National Science Foundation [DGE-0718123]
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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.
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