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Tadhg P. Begley - One of the best experts on this subject based on the ideXlab platform.
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The vitamin B1 metabolism of Staphylococcus aureus is controlled at enzymatic and transcriptional levels
PLoS ONE, 2009Co-Authors: Ingrid B. Müller, Bärbel Bergmann, Leonard Amaral, Isabel Couto, Matthew R Groves, Rolf D Walter, Tadhg P. Begley, Carsten WrengerAbstract:Vitamin B1 is in its active form Thiamine pyroPhosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and Thiamine Phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the Thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels.
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reconstitution of thic in Thiamine pyrimidine biosynthesis expands the radical sam superfamily
Nature Chemical Biology, 2008Co-Authors: Abhishek Chatterjee, Tadhg P. Begley, Yue Li, Y Zhang, Tyler L Grove, Carsten Krebs, Squire J Booker, S E EalickAbstract: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 (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C 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. Mossbauer 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.
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reconstitution of thic in Thiamine pyrimidine biosynthesis expands the radical sam superfamily
Nature Chemical Biology, 2008Co-Authors: Abhishek Chatterjee, Tadhg P. Begley, Y Zhang, Tyler L Grove, Carsten Krebs, Squire J Booker, Michael Lee, S E EalickAbstract: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 (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C 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. Mossbauer 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.
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Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine.
Biological chemistry, 2006Co-Authors: Carsten Wrenger, Ingrid B. Müller, Tadhg P. Begley, Marie-luise Eschbach, Nathan P. Laun, Rolf D WalterAbstract:Vitamin B1 (Thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and Thiamine Phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or Thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of Thiamine precursors.
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Characterization of the Bacillus subtilis thiC operon involved in Thiamine biosynthesis.
Journal of bacteriology, 1997Co-Authors: Yi Zhang, Sean V. Taylor, Hsiu-ju Chiu, Tadhg P. BegleyAbstract: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.
Pierre Wins - One of the best experts on this subject based on the ideXlab platform.
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Biochemistry of Thiamine and Thiamine Phosphate Compounds
Encyclopedia of Biological Chemistry, 2013Co-Authors: Lucien Bettendorff, Pierre WinsAbstract:Thiamine (vitamin B1) is an essential molecule for all living organisms. It is the precursor for several phosphorylated derivatives, the most important being the coenzyme Thiamine diPhosphate (ThDP). Plants and microorganisms synthesize Thiamine, but humans and other animals must rely on exogenous dietary sources. Thiamine is transported into cells by specific transporters and pyrophosphorylated to ThDP in the cytosol. ThDP is a cofactor for important catabolic reactions, that is, pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase (a limiting step in the Krebs cycle), branched-chain 2-oxo acid dehydrogenase, and transketolase (a key enzyme in the pentose Phosphate pathway). Therefore, ThDP is indispensable for oxidative energy metabolism. Humans are particularly sensitive to Thiamine deficiency, which mainly affects the nervous system and causes two classical diseases, beriberi (a polyneuritic syndrome) and Wernicke–Korsakoff syndrome (encephalopathy and dementia linked to alcoholism). The selective vulnerability of some brain regions is difficult to explain by general metabolic impairment; hence, a non-cofactor role of some Thiamine derivative(s) has been suggested. ThDP is the precursor for two triphosphorylated derivatives, Thiamine triPhosphate and adenosine Thiamine triPhosphate. In bacteria, those compounds are produced in response to specific conditions of nutritional downshift. Their possible role(s) in eukaryotic organisms are unknown.
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thiaminylated adenine nucleotides chemical synthesis structural characterization and natural occurrence
FEBS Journal, 2009Co-Authors: Michel Frederich, Tiziana Gigliobianco, Gabriel Mazzucchelli, Luc Angenot, Marjorie Gangolf, Edwin De Pauw, David Delvaux, Georges Dive, Benjamin Elias, Pierre WinsAbstract:Thiamine and its three phosphorylated derivatives (mono-, di- and triPhosphate) occur naturally in most cells. Recently, we reported the presence of a fourth Thiamine derivative, adenosine Thiamine triPhosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine Thiamine triPhosphate leads to another new compound, adenosine Thiamine diPhosphate, as a side product. The structure of both compounds was confirmed by MS analysis and H-1-, C-13- and P-31-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine Thiamine derivatives compared with conventional Thiamine Phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine Thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine Thiamine triPhosphate may account for 15% of the total Thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine Thiamine diPhosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural Thiamine adenine compounds further highlights the complexity and diversity of Thiamine biochemistry, which is not restricted to the cofactor role of Thiamine diPhosphate.
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Thiaminylated adenine nucleotides — chemical synthesis, structural characterization and natural occurrence
The FEBS journal, 2009Co-Authors: Michel Frederich, Tiziana Gigliobianco, Gabriel Mazzucchelli, Luc Angenot, Marjorie Gangolf, Edwin De Pauw, David Delvaux, Georges Dive, Benjamin Elias, Pierre WinsAbstract:Thiamine and its three phosphorylated derivatives (mono-, di- and triPhosphate) occur naturally in most cells. Recently, we reported the presence of a fourth Thiamine derivative, adenosine Thiamine triPhosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine Thiamine triPhosphate leads to another new compound, adenosine Thiamine diPhosphate, as a side product. The structure of both compounds was confirmed by MS analysis and H-1-, C-13- and P-31-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine Thiamine derivatives compared with conventional Thiamine Phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine Thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine Thiamine triPhosphate may account for 15% of the total Thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine Thiamine diPhosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural Thiamine adenine compounds further highlights the complexity and diversity of Thiamine biochemistry, which is not restricted to the cofactor role of Thiamine diPhosphate.
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Low Thiamine DiPhosphate Levels in Brains of Patients with Frontal Lobe Degeneration of the Non‐Alzheimer's Type
Journal of neurochemistry, 2002Co-Authors: Lucien Bettendorff, Pierre Wins, Frank Mastrogiacomo, Stephen J. Kish, Thierry Grisar, Melvyn J. BallAbstract:We compared the Thiamine and Thiamine Phosphate contents in the frontal, temporal, parietal, and occipital cortex of six patients with frontal lobe degeneration of the non-Alzheimer's type (FNAD) or frontotemporal dementia with five age-, postmortem delay-, and agonal status-matched control subjects. Our results reveal a 40-50% decrease in Thiamine diPhosphate (TDP) in the cortex of FNAD patients, whereas Thiamine monoPhosphate was increased 49-119%. TDP synthesizing and hydrolyzing enzymes were unaffected. The activity of citrate synthase, a mitochondrial marker enzyme, was decreased in the frontal cortex of patients with FNAD, but no correlation with TDP content was found. These results suggest that decreased contents of TDP, which is essentially mitochondrial, is a specific feature of FNAD. As TDP is an essential cofactor for oxidative metabolism and neurotransmitter synthesis, and because low Thiamine status (compared with other species) is a constant feature in humans, a nearly 50% decrease in cortical TDP content may contribute significantly to the clinical symptoms observed in FNAD. This study also provides a basis for a trial of Thiamine, to improve the cognitive status of the patients.
S E Ealick - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of thic in Thiamine pyrimidine biosynthesis expands the radical sam superfamily
Nature Chemical Biology, 2008Co-Authors: Abhishek Chatterjee, Tadhg P. Begley, Yue Li, Y Zhang, Tyler L Grove, Carsten Krebs, Squire J Booker, S E EalickAbstract: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 (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C 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. Mossbauer 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.
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reconstitution of thic in Thiamine pyrimidine biosynthesis expands the radical sam superfamily
Nature Chemical Biology, 2008Co-Authors: Abhishek Chatterjee, Tadhg P. Begley, Y Zhang, Tyler L Grove, Carsten Krebs, Squire J Booker, Michael Lee, S E EalickAbstract: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 (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C 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. Mossbauer 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.
Lucien Bettendorff - One of the best experts on this subject based on the ideXlab platform.
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Biochemistry of Thiamine and Thiamine Phosphate Compounds
Encyclopedia of Biological Chemistry, 2013Co-Authors: Lucien Bettendorff, Pierre WinsAbstract:Thiamine (vitamin B1) is an essential molecule for all living organisms. It is the precursor for several phosphorylated derivatives, the most important being the coenzyme Thiamine diPhosphate (ThDP). Plants and microorganisms synthesize Thiamine, but humans and other animals must rely on exogenous dietary sources. Thiamine is transported into cells by specific transporters and pyrophosphorylated to ThDP in the cytosol. ThDP is a cofactor for important catabolic reactions, that is, pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase (a limiting step in the Krebs cycle), branched-chain 2-oxo acid dehydrogenase, and transketolase (a key enzyme in the pentose Phosphate pathway). Therefore, ThDP is indispensable for oxidative energy metabolism. Humans are particularly sensitive to Thiamine deficiency, which mainly affects the nervous system and causes two classical diseases, beriberi (a polyneuritic syndrome) and Wernicke–Korsakoff syndrome (encephalopathy and dementia linked to alcoholism). The selective vulnerability of some brain regions is difficult to explain by general metabolic impairment; hence, a non-cofactor role of some Thiamine derivative(s) has been suggested. ThDP is the precursor for two triphosphorylated derivatives, Thiamine triPhosphate and adenosine Thiamine triPhosphate. In bacteria, those compounds are produced in response to specific conditions of nutritional downshift. Their possible role(s) in eukaryotic organisms are unknown.
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benfotiamine a synthetic s acyl Thiamine derivative has different mechanisms of action and a different pharmacological profile than lipid soluble Thiamine disulfide derivatives
BMC Pharmacology, 2008Co-Authors: Marielaure Volvert, Sandrine Seyen, Marie Piette, Brigitte Evrard, Marjorie Gangolf, Jean Christophe Plumier, Lucien BettendorffAbstract:Background Lipid-soluble Thiamine precursors have a much higher bioavailability than genuine Thiamine and therefore are more suitable for therapeutic purposes. Benfotiamine (S-benzoylThiamine O-monoPhosphate), an amphiphilic S-acyl Thiamine derivative, prevents the progression of diabetic complications, probably by increasing tissue levels of Thiamine diPhosphate and so enhancing transketolase activity. As the brain is particularly sensitive to Thiamine deficiency, we wanted to test whether intracellular Thiamine and Thiamine Phosphate levels are increased in the brain after oral benfotiamine administration.
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Benfotiamine, a synthetic S-acyl Thiamine derivative, has different mechanisms of action and a different pharmacological profile than lipid-soluble Thiamine disulfide derivatives
BMC Pharmacology, 2008Co-Authors: Marielaure Volvert, Sandrine Seyen, Marie Piette, Brigitte Evrard, Marjorie Gangolf, Jean Christophe Plumier, Lucien BettendorffAbstract:Background Lipid-soluble Thiamine precursors have a much higher bioavailability than genuine Thiamine and therefore are more suitable for therapeutic purposes. Benfotiamine (S-benzoylThiamine O-monoPhosphate), an amphiphilic S-acyl Thiamine derivative, prevents the progression of diabetic complications, probably by increasing tissue levels of Thiamine diPhosphate and so enhancing transketolase activity. As the brain is particularly sensitive to Thiamine deficiency, we wanted to test whether intracellular Thiamine and Thiamine Phosphate levels are increased in the brain after oral benfotiamine administration. Results Benfotiamine that is practically insoluble in water, organic solvents or oil was solubilized in 200 mM hydroxypropyl-β-cyclodextrin and the mice received a single oral administration of 100 mg/kg. Though Thiamine levels rapidly increased in blood and liver to reach a maximum after one or two hours, no significant increase was observed in the brain. When mice received a daily oral administration of benfotiamine for 14 days, Thiamine derivatives were increased significantly in the liver but not in the brain, compared to control mice. In addition, incubation of cultured neuroblastoma cells with 10 μM benfotiamine did not lead to increased intracellular Thiamine levels. Moreover, in Thiamine-depleted neuroblastoma cells, intracellular Thiamine contents increased more rapidly after addition of Thiamine to the culture medium than after addition of benfotiamine for which a lag period was observed. Conclusion Our results show that, though benfotiamine strongly increases Thiamine levels in blood and liver, it has no significant effect in the brain. This would explain why beneficial effects of benfotiamine have only been observed in peripheral tissues, while sulbutiamine, a lipid-soluble Thiamine disulfide derivative, that increases Thiamine derivatives in the brain as well as in cultured cells, acts as a central nervous system drug. We propose that benfotiamine only penetrates the cells after dephosphorylation by intestinal alkaline phosphatases. It then enters the bloodstream as S-benzoylThiamine that is converted to Thiamine in erythrocytes and in the liver. Benfotiamine, an S-acyl derivative practically insoluble in organic solvents, should therefore be differentiated from truly lipid-soluble Thiamine disulfide derivatives (alliThiamine and the synthetic sulbutiamine and fursultiamine) with a different mechanism of absorption and different pharmacological properties.
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Thiamine, Thiamine Phosphates, and their metabolizing enzymes in human brain.
Journal of neurochemistry, 2002Co-Authors: Lucien Bettendorff, Frank Mastrogiacomo, Stephen J. Kish, Thierry GrisarAbstract:Total Thiamine (the sum of Thiamine and its Phosphate esters) concentrations are two- to fourfold lower in human brain than in the brain of other mammals. There were no differences in the total Thiamine content between biopsied and autopsied human brain, except that in the latter, Thiamine triPhosphate was undetectable. The main Thiamine Phosphate-metabolizing enzymes could be detected in autopsied brain, and the kinetic parameters were comparable to those reported in other species. Thiamine diPhosphate levels were lowest in hippocampus (15 +/- 4 pmol/mg of protein) and highest in mammillary bodies (24 +/- 4 pmol/mg of protein). Maximal levels of Thiamine and its Phosphate ester were found to be present at birth. In parietal cortex and globus pallidus, mean levels of total Thiamine in the oldest age group (77-103 years) were, respectively, 21 and 26% lower than those in the middle age group (40-55 years). Unlike cerebral cortex, the globus pallidus showed a sharp drop in Thiamine diPhosphate levels during infancy, with concentrations in the oldest group being only approximately 50% of the levels present during the first 4 months of life. These data, consistent with previous observations conducted in blood, suggest a tendency toward decreased Thiamine status in older people.
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Low Thiamine DiPhosphate Levels in Brains of Patients with Frontal Lobe Degeneration of the Non‐Alzheimer's Type
Journal of neurochemistry, 2002Co-Authors: Lucien Bettendorff, Pierre Wins, Frank Mastrogiacomo, Stephen J. Kish, Thierry Grisar, Melvyn J. BallAbstract:We compared the Thiamine and Thiamine Phosphate contents in the frontal, temporal, parietal, and occipital cortex of six patients with frontal lobe degeneration of the non-Alzheimer's type (FNAD) or frontotemporal dementia with five age-, postmortem delay-, and agonal status-matched control subjects. Our results reveal a 40-50% decrease in Thiamine diPhosphate (TDP) in the cortex of FNAD patients, whereas Thiamine monoPhosphate was increased 49-119%. TDP synthesizing and hydrolyzing enzymes were unaffected. The activity of citrate synthase, a mitochondrial marker enzyme, was decreased in the frontal cortex of patients with FNAD, but no correlation with TDP content was found. These results suggest that decreased contents of TDP, which is essentially mitochondrial, is a specific feature of FNAD. As TDP is an essential cofactor for oxidative metabolism and neurotransmitter synthesis, and because low Thiamine status (compared with other species) is a constant feature in humans, a nearly 50% decrease in cortical TDP content may contribute significantly to the clinical symptoms observed in FNAD. This study also provides a basis for a trial of Thiamine, to improve the cognitive status of the patients.
Abhishek Chatterjee - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of thic in Thiamine pyrimidine biosynthesis expands the radical sam superfamily
Nature Chemical Biology, 2008Co-Authors: Abhishek Chatterjee, Tadhg P. Begley, Yue Li, Y Zhang, Tyler L Grove, Carsten Krebs, Squire J Booker, S E EalickAbstract: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 (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C 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. Mossbauer 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.
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reconstitution of thic in Thiamine pyrimidine biosynthesis expands the radical sam superfamily
Nature Chemical Biology, 2008Co-Authors: Abhishek Chatterjee, Tadhg P. Begley, Y Zhang, Tyler L Grove, Carsten Krebs, Squire J Booker, Michael Lee, S E EalickAbstract: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 (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C 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. Mossbauer 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.