Publications

• A th-1 mutant of Arabidopsis thaliana is defective for a thiamin-phosphate-synthesizing enzyme: thiamin phosphate pyrophosphorylase.

  Komeda Y., Tanaka M., Nishimune T.

  We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 x 10(-7) molar thiamin were used for the examination of the production of thiamin an ... >> More

  We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 x 10(-7) molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45 degrees C, and the K(m) values for the substrates are 2.7 x 10(-6) molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 x 10(-6) molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate. << Less

  Plant Physiol. 88:248-250(1988) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The biosynthesis of thiamine. 2. Fractionation of enzyme system and identification of thiazole monophosphate and thiamine monophosphate as intermediates.

  CAMIENER G.W., BROWN G.M.

  J Biol Chem 235:2411-2417(1960) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Characterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis.

  Zhang Y., Taylor S.V., Chiu H.-J., Begley T.P.

  The characterization of a three-gene operon (the thiC operon) at 331 min, which is involved in thiamine biosynthesis in Bacillus subtilis, is described. The first gene in the operon is homologous to transcription activators in the lysR family. The second and third genes (thiK and thiC) have been s ... >> More

  The characterization of a three-gene operon (the thiC operon) at 331 min, which is involved in thiamine biosynthesis in Bacillus subtilis, is described. The first gene in the operon is homologous to transcription activators in the lysR family. The second and third genes (thiK and thiC) have been subcloned and overexpressed in Escherichia coli. ThiK (30 kDa) catalyzes the phosphorylation of 4-methyl-5-(beta-hydroxyethyl)thiazole. ThiC (27 kDa) catalyzes the substitution of the pyrophosphate of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate to yield thiamine phosphate. Transcription of the thiC operon is not regulated by thiamine or 2-methyl-4-amino-5-hydroxymethylpyrimidine and is only slightly repressed by 4-methyl-5-(beta-hydroxyethyl)thiazole. << Less

  J. Bacteriol. 179:3030-3035(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The enzymatic synthesis of thiamine monophosphate.

  LEDER I.G.

  J Biol Chem 236:3066-3071(1961) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1.

  Ajjawi I., Tsegaye Y., Shintani D.

  2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus prov ... >> More

  2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus proving that the th1 locus corresponds to the structural gene for the HMPPK/TMPPase. Sequence comparisons between the wild-type HMPPK/TMPPase gene and the th1-201 mutant allele identified a single point mutation that caused the substitution of a phenylalanine for a conserved serine residue in the HMPPK domain. Functional analyses of the mutant HMPPK/TMPPase in Escherichia coli revealed that the amino acid substitution in the HMPPK domain of mutant enzyme resulted in a conformational change that severely compromised both activities of the bifunctional enzyme. Studies were also performed to identify the chloroplast as the specific subcellular locale of the Arabidopsis HMPPK/TMPPase. << Less

  Arch. Biochem. Biophys. 459:107-114(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

  Kim Y.S., Nosaka K., Downs D.M., Kwak J.M., Park D., Chung I.K., Nam H.G.

  We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The ... >> More

  We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene. << Less

  Plant Mol. Biol. 37:955-966(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Systematic discovery of analogous enzymes in thiamin biosynthesis.

  Morett E., Korbel J.O., Rajan E., Saab-Rincon G., Olvera L., Olvera M., Schmidt S., Snel B., Bork P.

  In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species. In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequenc ... >> More

  In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species. In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequence and structural similarity. Here we fill such gaps by analyzing anticorrelating occurrences of genes across species. Our approach, applied to the thiamin biosynthesis pathway comprising approximately 15 catalytic steps, predicts seven instances in which known enzymes have been displaced by analogous proteins. So far we have verified four predictions by genetic complementation, including three proteins for which there was no previous experimental evidence of a role in the thiamin biosynthesis pathway. For one hypothetical protein, biochemical characterization confirmed the predicted thiamin phosphate synthase (ThiE) activity. The results demonstrate the ability of our computational approach to predict specific functions without taking into account sequence similarity. << Less

  Nat. Biotechnol. 21:790-795(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs.

  Khare G., Kar R., Tyagi A.K.

  Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidr ... >> More

  Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M. tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M. tuberculosis. In this study, a comparative homology model of M. tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC(50) values ranging from 20-100 µg/ml and two of these exhibited weak inhibition of M. tuberculosis growth with MIC(99) values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M. tuberculosis growth with an MIC(99) value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M. tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis. << Less

  PLoS ONE 6:E22441-E22441(2011) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.

  Chiu H.-J., Reddick J.J., Begley T.P., Ealick S.E.

  The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/be ... >> More

  The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme. << Less

  Biochemistry 38:6460-6470(1999) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway.

  Kawasaki Y.

  Mutants of Saccharomyces cerevisiae resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole, an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), which are deficient in the activities of both hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase, invol ... >> More

  Mutants of Saccharomyces cerevisiae resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole, an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), which are deficient in the activities of both hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase, involved in the pathway of de novo synthesis of thiamine in S. cerevisiae, have been isolated. Genetic analysis revealed that the mutation occurs at a single gene in the nucleus. The two enzyme activities were copurified to apparent homogeneity, and the molecular masses of the purified proteins were found to be approximately 470 and 60 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Hydroxyethylthiazole kinase was specific for ATP and Mg2+, although to a lesser extent a combination with other nucleoside triphosphates or divalent cations could replace them. p-Chloromercuribenzoate was a potent inhibitor of the enzyme, and the inhibition was prevented by the addition of 2-mercaptoethanol. These findings indicate that yeast hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity, which is an octamer of identical 60-kDa subunits. << Less

  J Bacteriol 175:5153-5158(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.