11 results on '"Thiamine chemistry"'
Search Results
2. The last piece in the vitamin B1 biosynthesis puzzle: structural and functional insight into yeast 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase.
- Author
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Coquille S, Roux C, Fitzpatrick TB, and Thore S
- Subjects
- Crystallography, X-Ray, Iron metabolism, Mutation, Phosphotransferases (Phosphate Group Acceptor) genetics, Phosphotransferases (Phosphate Group Acceptor) metabolism, Protein Binding, Protein Structure, Tertiary, Pyrimidines chemistry, Pyrimidines metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Structure-Activity Relationship, Thiamine chemistry, Thiamine genetics, Iron chemistry, Phosphotransferases (Phosphate Group Acceptor) chemistry, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins chemistry, Thiamine biosynthesis
- Abstract
Vitamin B(1) is essential for all organisms being well recognized as a necessary cofactor for key metabolic pathways such as glycolysis, and was more recently implicated in DNA damage responses. Little is known about the enzyme responsible for the formation of the pyrimidine moiety (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase). We report a structure-function study of the HMP-P synthase from yeast, THI5p. Our crystallographic structure shows that THI5p is a mix between periplasmic binding proteins and pyridoxal 5'-phosphate-dependent enzymes. Mutational and yeast complementation studies identify the key residues for HMP-P biosynthesis as well as the use of pyridoxal 5'-phosphate as a substrate rather than as a cofactor. Furthermore, we could show that iron binding to HMP-P synthase is essential for the reaction.
- Published
- 2012
- Full Text
- View/download PDF
3. Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a "direct pathway" between the 4'-aminopyrimidine N1' atoms.
- Author
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Nemeria NS, Arjunan P, Chandrasekhar K, Mossad M, Tittmann K, Furey W, and Jordan F
- Subjects
- Catalysis, Circular Dichroism, Crystallography, X-Ray methods, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Binding, Pyruvate Dehydrogenase (Lipoamide) chemistry, Thiamine Pyrophosphate chemistry, Vitamin B Complex chemistry, 4-Aminopyridine chemistry, Escherichia coli metabolism, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Pyruvate Dehydrogenase (Lipoamide) metabolism, Thiamine chemistry
- Abstract
Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4'-aminopyrimidine N1' atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu(571), Glu(235), and Glu(237)) and Arg(606) resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu(235) makes no direct contact with the cofactor. The role of the conserved Glu(571) residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.
- Published
- 2010
- Full Text
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4. Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro.
- Author
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Kriek M, Martins F, Leonardi R, Fairhurst SA, Lowe DJ, and Roach PL
- Subjects
- Escherichia coli Proteins genetics, Escherichia coli Proteins isolation & purification, Molecular Structure, Nucleotidyltransferases genetics, Nucleotidyltransferases metabolism, Protein Structure, Quaternary, Protein Subunits genetics, Protein Subunits isolation & purification, Thiamine biosynthesis, Thiamine chemistry, Thiazoles chemistry, Tyrosine metabolism, Escherichia coli enzymology, Escherichia coli Proteins metabolism, Protein Subunits metabolism, Thiazoles metabolism
- Abstract
Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction.
- Published
- 2007
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5. Targeting and trafficking of the human thiamine transporter-2 in epithelial cells.
- Author
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Subramanian VS, Marchant JS, and Said HM
- Subjects
- Animals, Bacterial Proteins metabolism, Caco-2 Cells, Cell Line, Cell Line, Tumor, Cell Membrane metabolism, Cytoplasm metabolism, DNA Mutational Analysis, DNA Primers chemistry, DNA, Complementary metabolism, Dogs, Dynactin Complex, Flow Cytometry, Green Fluorescent Proteins chemistry, Humans, Kidney metabolism, Luminescent Proteins metabolism, Microscopy, Confocal, Microscopy, Fluorescence, Microscopy, Video, Microtubule-Associated Proteins chemistry, Microtubules chemistry, Microtubules metabolism, Multigene Family, Mutation, Peptides chemistry, Polymerase Chain Reaction, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Recombinant Fusion Proteins chemistry, Thiamine chemistry, Transfection, Epithelial Cells metabolism, Membrane Transport Proteins metabolism
- Abstract
Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.
- Published
- 2006
- Full Text
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6. Restoration of transport activity by co-expression of human reduced folate carrier half-molecules in transport-impaired K562 cells: localization of a substrate binding domain to transmembrane domains 7-12.
- Author
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Witt TL, Stapels SE, and Matherly LH
- Subjects
- Amino Acid Sequence, Biological Transport, Cell Line, Cell Membrane metabolism, Dose-Response Relationship, Drug, Epitopes chemistry, Folic Acid chemistry, Folic Acid Antagonists pharmacology, Glycosylation, Humans, Immunoprecipitation, K562 Cells, Kinetics, Leucovorin pharmacology, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase pharmacology, Methotrexate pharmacology, Microscopy, Confocal, Molecular Sequence Data, Peptides chemistry, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Reduced Folate Carrier Protein, Sodium Dodecyl Sulfate chemistry, Substrate Specificity, Thiamine chemistry, Time Factors, Transfection, Tunicamycin pharmacology, Membrane Transport Proteins metabolism, Methotrexate analogs & derivatives
- Abstract
Reduced folates such as 5-methyl tetrahydrofolate and classical antifolates such as methotrexate are actively transported into mammalian cells by the reduced folate carrier (RFC). RFC is characterized by 12 stretches of mostly hydrophobic, alpha-helix-promoting amino acids, internally oriented N and C termini, and a large central linker connecting transmembrane domains (TMDs) 1-6 and 7-12. Previous studies showed that deletion of the majority of the central loop domain between TMDs 6 and 7 abolished transport, but this segment could be replaced with mostly non-homologous sequence from the SLC19A2 thiamine transporter to restore transport function. In this report, we expressed RFC from separate TMD1-6 and TMD7-12 RFC half-molecule constructs, each with a unique epitope tag, in RFC-null K562 cells to restore transport activity. Restored transport exhibited characteristic transport kinetics for methotrexate, a capacity for trans-stimulation by pretreatment with leucovorin, and inhibition by N-hydroxysuccinimide methotrexate, a documented affinity inhibitor of RFC. The TMD1-6 half-molecule migrated on SDS gels as a 38-58 kDa glycosylated species and was converted to 27 kDa by N-glycosidase F or tunicamycin treatments. The 40 kDa TMD7-12 half-molecule was unaffected by these treatments. Using transfected cells expressing both TMDs 1-6 and TMDs 7-12 as separate polypeptides, the TMD7-12 half-molecule was covalently radiolabeled with N-hydroxysuccinimide [(3)H]methotrexate. No radioactivity was incorporated into the TMD1-6 half-molecule. Digestion with endoproteinase GluC decreased the size of the radiolabeled 40 kDa TMD7-12 polypeptide to approximately 20 kDa. Our results demonstrate that a functional RFC can be reconstituted with RFC half-molecules and localize a critical substrate binding domain to within TMDs 7-12.
- Published
- 2004
- Full Text
- View/download PDF
7. Structural and biochemical basis for novel mutations in homozygous Israeli maple syrup urine disease patients: a proposed mechanism for the thiamin-responsive phenotype.
- Author
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Chuang JL, Wynn RM, Moss CC, Song JL, Li J, Awad N, Mandel H, and Chuang DT
- Subjects
- Alleles, Blotting, Western, Centrifugation, Density Gradient, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Humans, Israel, Kinetics, Methylamines pharmacology, Models, Chemical, Models, Molecular, Phenotype, Protein Binding, Protein Folding, Protein Structure, Tertiary, Recombinant Proteins metabolism, Thermodynamics, Thiamine chemistry, Thiamine Pyrophosphate chemistry, Homozygote, Maple Syrup Urine Disease genetics, Mutation
- Abstract
Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-alpha (TGC to TGG) in the E1alpha subunit; H156Y-beta (CAT to TAT), V69G-beta (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1beta subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-alpha or H156Y-beta mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-beta substitution cannot be expressed, suggesting aberrant folding caused by this mutation. These E1 mutations are ubiquitously associated with the classic phenotype in homozygous-affected patients. The H391R substitution in the E2 subunit abolishes the key catalytic residue that functions as a general base in the acyltransfer reaction, resulting in a completely inactive E2 component. However, wild-type E1 activity is enhanced by E1 binding to this full-length mutant E2 in vitro. We propose that the augmented E1 activity is responsible for robust thiamin responsiveness in homozygous patients carrying the H391R E2 mutation and that the presence of a full-length mutant E2 is diagnostic of this MSUD phenotype. The present results offer a structural and biochemical basis for these novel mutations and will facilitate DNA-based diagnosis for MSUD in the Israeli population.
- Published
- 2004
- Full Text
- View/download PDF
8. Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity.
- Author
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Leonardi R and Roach PL
- Subjects
- Adenosine metabolism, Carbon-Sulfur Lyases physiology, Carrier Proteins metabolism, Cell-Free System, Chromatography, High Pressure Liquid, Culture Media metabolism, Cysteine chemistry, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Escherichia coli Proteins metabolism, Ligases chemistry, Models, Chemical, NADP metabolism, Nucleotidyltransferases metabolism, Plasmids metabolism, S-Adenosylmethionine metabolism, Sulfurtransferases physiology, Thiamine chemistry, Time Factors, Tyrosine metabolism, Tyrosine physiology, Bacterial Proteins, Carrier Proteins chemistry, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Nucleotidyltransferases chemistry, Thiamine analogs & derivatives, Thiamine biosynthesis, Thiazoles chemistry
- Abstract
The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI. We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E. coli 83-1 cells. The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E. coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells. The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH.
- Published
- 2004
- Full Text
- View/download PDF
9. Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition.
- Author
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Nemeria N, Yan Y, Zhang Z, Brown AM, Arjunan P, Furey W, Guest JR, and Jordan F
- Subjects
- Amino Acid Sequence, Base Sequence, Circular Dichroism, DNA Primers, Enzyme Inhibitors chemistry, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Pyruvate Dehydrogenase Complex chemistry, Pyruvate Dehydrogenase Complex genetics, Pyruvate Dehydrogenase Complex metabolism, Sequence Homology, Amino Acid, Spectrometry, Fluorescence, Thiamine chemistry, Thiazoles chemistry, Enzyme Inhibitors pharmacology, Escherichia coli enzymology, Pyruvate Dehydrogenase Complex antagonists & inhibitors, Thiazoles pharmacology, Tyrosine metabolism
- Abstract
Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.
- Published
- 2001
- Full Text
- View/download PDF
10. The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA.
- Author
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Mueller EG, Palenchar PM, and Buck CJ
- Subjects
- Alanine chemistry, Amino Acid Motifs, Carbon-Sulfur Lyases metabolism, Cloning, Molecular, Disulfides chemistry, Dithionitrobenzoic Acid pharmacology, Escherichia coli metabolism, Iron chemistry, Models, Chemical, Mutagenesis, Site-Directed, Plasmids metabolism, Spectrophotometry, Sulfur chemistry, Thiamine chemistry, Time Factors, Ultraviolet Rays, Uracil chemistry, Bacterial Proteins, Cysteine chemistry, Cysteine physiology, Escherichia coli Proteins, RNA, Transfer chemistry, Sulfurtransferases chemistry, Thiouridine chemistry
- Abstract
The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283-8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.
- Published
- 2001
- Full Text
- View/download PDF
11. The synthesis of benzyl-(3)-thiazolium chloride analogues of thiamine.
- Author
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LIVERMORE AH and SEALOCK RR
- Subjects
- Chlorides, Ions, Organic Chemicals, Thiamine chemistry
- Published
- 1947
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