Publications

• Biosynthesis of the thiamin pyrimidine: the reconstitution of a remarkable rearrangement reaction.

  Lawhorn B.G., Mehl R.A., Begley T.P.

  The conversion of 5-aminoimidazole ribonucleotide (AIR) into 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) is a fascinating reaction on the thiamin biosynthetic pathway in bacteria and is probably the most complex unresolved rearrangement in primary metabolism. We have successfully reconstitute ... >> More

  The conversion of 5-aminoimidazole ribonucleotide (AIR) into 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) is a fascinating reaction on the thiamin biosynthetic pathway in bacteria and is probably the most complex unresolved rearrangement in primary metabolism. We have successfully reconstituted this reaction in a cell-free system. The E. coli thiC gene product and an additional unidentified E. coli protein are required for the reaction. In addition, SAM and nicotinamide cofactors are required for full activity. Labeling studies to determine the origin of most of the atoms in the pyrimidine are described. Based on these studies, a new mechanism for HMP formation is proposed. << Less

  Org. Biomol. Chem. 2:2538-2546(2004) [PubMed] [EuropePMC]

• ThiC is an [Fe-S] cluster protein that requires AdoMet to generate the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety in thiamin synthesis.

  Martinez-Gomez N.C., Downs D.M.

  Thiamin pyrophosphate is a required cofactor in all organisms. The biosynthesis of thiamin requires the independently synthesized 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) and 5-hydroxyethyl-4-methylthiazole phosphate (THZ-P) moieties. In bacteria, the pyrimidine moiety is ... >> More

  Thiamin pyrophosphate is a required cofactor in all organisms. The biosynthesis of thiamin requires the independently synthesized 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) and 5-hydroxyethyl-4-methylthiazole phosphate (THZ-P) moieties. In bacteria, the pyrimidine moiety is derived from 5-aminoimidazole ribotide (AIR), and ThiC is the only gene product known to be required for this conversion in vivo. We report here the purification and characterization of the ThiC protein from Salmonella enterica. The data showed this protein generated HMP when AIR, S-adenosylmethionine (AdoMet), and an appropriate reducing agent were present. It is further shown that ThiC carries an oxygen labile [Fe-S] cluster essential for this activity. << Less

  Biochemistry 47:9054-9056(2008) [PubMed] [EuropePMC]

• Reaction of AdoMet with ThiC generates a backbone free radical.

  Martinez-Gomez N.C., Poyner R.R., Mansoorabadi S.O., Reed G.H., Downs D.M.

  ThiC is an [4Fe-4S] cluster protein that catalyzes the formation of 4-amino-5-hydroxymethyl-2-methylpyrimidine. EPR spectroscopic studies demonstrate that, upon interaction with AdoMet, active ThiC from Salmonella enterica generates a persistent free radical on the alpha-carbon of an amino acid re ... >> More

  ThiC is an [4Fe-4S] cluster protein that catalyzes the formation of 4-amino-5-hydroxymethyl-2-methylpyrimidine. EPR spectroscopic studies demonstrate that, upon interaction with AdoMet, active ThiC from Salmonella enterica generates a persistent free radical on the alpha-carbon of an amino acid residue. The EPR properties of the radical are consistent with any residue other than a Gly or Ala. Exposure to oxygen was accompanied by a fission of the radical-carrying polypeptide chain between the Gly436 and His437 residues in ThiC. Regardless of whether the backbone radical is part of the catalytic machinery, its presence provides evidence that ThiC employs free radical chemistry as expected for radical SAM enzymes. << Less

  Biochemistry 48:217-219(2009) [PubMed] [EuropePMC]

• A 'radical dance' in thiamin biosynthesis: mechanistic analysis of the bacterial hydroxymethylpyrimidine phosphate synthase.

  Chatterjee A., Hazra A.B., Abdelwahed S., Hilmey D.G., Begley T.P.

  Angew. Chem. Int. Ed. 49:8653-8656(2010) [PubMed] [EuropePMC]

• Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily.

  Chatterjee A., Li Y., Zhang Y., Grove T.L., Lee M., Krebs C., Booker S.J., Begley T.P., Ealick S.E.

  4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the p ... >> More

  4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the product HMP-P and a substrate analog imidazole ribotide. The structure of HMP-P synthase reveals a homodimer in which each protomer comprises three domains: an N-terminal domain with a novel fold, a central (betaalpha)(8) barrel and a disordered C-terminal domain that contains a conserved CX(2)CX(4)C motif, which is suggestive of a [4Fe-4S] cluster. Biochemical studies have confirmed that HMP-P synthase is iron sulfur cluster-dependent, that it is a new member of the radical SAM superfamily and that HMP-P and 5'-deoxyadenosine are products of the reaction. Mössbauer and EPR spectroscopy confirm the presence of one [4Fe-4S] cluster. Structural comparisons reveal that HMP-P synthase is homologous to a group of adenosylcobalamin radical enzymes. This similarity supports an evolutionary relationship between these two superfamilies. << Less

  Nat. Chem. Biol. 4:758-765(2008) [PubMed] [EuropePMC]

• A connection between iron-sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica.

  Dougherty M.J., Downs D.M.

  Several cellular pathways have been identified which affect the efficiency of thiamine biosynthesis in Salmonella enterica. Mutants defective in iron-sulfur (Fe-S) cluster metabolism are less efficient at synthesis of the pyrimidine moiety of thiamine. These mutants are compromised for the convers ... >> More

  Several cellular pathways have been identified which affect the efficiency of thiamine biosynthesis in Salmonella enterica. Mutants defective in iron-sulfur (Fe-S) cluster metabolism are less efficient at synthesis of the pyrimidine moiety of thiamine. These mutants are compromised for the conversion of aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), not the synthesis of AIR. The gene product ThiC contains potential ligands for an Fe-S cluster that are required for function in vivo. The conversion of AIR to HMP-P is sensitive to oxidative stress, and variants of ThiC have been identified that have increased sensitivity to oxidative growth conditions. The data are consistent with ThiC or an as-yet-unidentified protein involved in HMP-P synthesis containing an Fe-S cluster required for its physiological function. << Less

  Microbiology 152:2345-2353(2006) [PubMed] [EuropePMC]