The control mechanism of thiamine biosynthesis a model for the study of control of converging pathways

Newell, P. C.; Tucker, R. G.

doi:10.1042/bj1000517

Cited by 37 publications

(15 citation statements)

References 5 publications

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“…We also noted that under conditions where thiI mutants were grown with limiting thiamine, HMP was excreted into the growth medium (24). This result was consistent with previous data suggesting that HMP does not feedback inhibit its own synthesis (19) and supported a role for thiI in thiazole synthesis.…”

supporting

confidence: 93%

Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium

1997

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Thiamine pyrophosphate (TPP) is a required cofactor in Salmonella typhimurium that is generated de novo by the condensation of 4-amino-5-hydroxymethyl pyrimidine (HMP) pyrophosphate and 4-methyl-5-(␤-hydroxyethyl)-thiazole (THZ) monophosphate. The THZ and HMP moieties are independently synthesized, and labeling studies have demonstrated probable metabolic precursors to both. We present herein the initial characterization of thiI, a gene required for THZ synthesis. We show that thiI is a 1,449-bp open reading frame located at minute 9.65 on the S. typhimurium chromosome and that it encodes a 483-amino-acid protein with a predicted molecular mass of 55 kDa. Unlike genes in the thiamine biosynthetic operon at minute 90, thiI is not transcriptionally regulated by TPP.Thiamine pyrophosphate (TPP) is a required cofactor for a number of well-characterized enzymes (e.g., ␣-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and transketolase). Although several recent studies of Saccharomyces cerevisiae (20-23), Escherichia coli (2, 28), and Salmonella typhimurium (9, 25, 29) have furthered our understanding of the synthesis of this vitamin, the genetic and biochemical characterization of the biosynthetic pathway is still in the early stages. In S. typhimurium and E. coli, thiamine monophosphate (TMP) is generated by the condensation of 4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) and 4-methyl-5-(␤-hydroxyethyl)-thiazole monophosphate (THZ-P) and then phosphorylated to TPP, the physiologically relevant form of the cofactor (Fig.

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supporting

confidence: 93%

Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium

1997

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“…another system for controlling Thz-P biosynthesis and indicate that it does not operate at the transcriptional or translational level (43), although the precise mechanism has yet to be characterized. The in vitro assays for thiazole formation contained an excess of the reagents and enzymes required to convert Thz-P to TP, thus ensuring that this step was never rate-limiting (and control experiments showed that this was the case).…”

Section: Reaction Of Radiochemically Labeled Thih Substrates-the Actimentioning

confidence: 99%

Thiazole Synthase from Escherichia coli

Kriek

Martins

Leonardi

et al. 2007

Journal of Biological Chemistry

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Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(␤-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 phosphateforming 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.Thiamine (vitamin B 1 ) is a key nutrient for humans with a recommended daily dose of 1.4 mg, and a deficiency of thiamine causes the disease beriberi (1). Thiamine pyrophosphate (TPP) 3 is an essential cofactor for several enzymes involved in carbohydrate and amino acid metabolism, including transketolase, pyruvate dehydrogenase, and ␣-ketoglutarate dehydrogenase (2, 3). Although studied for many years, the biosynthetic steps leading to TPP are not fully understood. Thiamine phosphate (TP) (Fig. 1, 6) is formed by ThiE, an enzyme that covalently links two independently formed heterocyclic precursors, 4-amino-5-hydroxymethylpyrimidine-pyrophosphate (Fig. 1, 5, Hmp-PP) and 4-methyl-5-(␤-hydroxyethyl)-thiazole phosphate (Thz-P) (Fig. 1, 4). This is converted to TPP by a ThiLdependent phosphorylation reaction (4, 5). 4-Amino-5-hydroxymethylpyrimidine phosphate is formed by a complex rearrangement of 5-aminoimidazole ribotide by ThiC (6), which upon phosphorylation by ThiD results in 4-amino-5-hydroxymethylpyrimidine-pyrophosphate (6, 7).The synthesis of Thz-P has been studied in a number of prokaryotes, most notably in the facultative anaerobes Escherichia coli and Salmonella sp., the aerobe Bacillus subtilis, and the eukaryote Saccharomyces cerevisiae (8, 9). Whole cell feeding studies in E. coli and knock-out mutants thereof indicated that Thz-P synthesis is dependent on th...

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“…The position of AICAR in the purine pathway indicated its effect on thiamine synthesis was indirect. Past workers suggested that the site of this effect was PurF, which was shown to be inhibited allosterically by a number of purine nucleotides (32,35,36). In vitro studies with purified PurF (33,52,59) have not directly addressed an allosteric role for AICAR (H. Zalkin, personal communication).…”

Section: Thimentioning

confidence: 99%

Metabolic Flux in Both the Purine Mononucleotide and Histidine Biosynthetic Pathways Can Influence Synthesis of the Hydroxymethyl Pyrimidine Moiety of Thiamine in Salmonella enterica

2002

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Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.Cellular metabolism requires the control of flux through a complex network of pathways. This control is accomplished by regulation at several levels. Regulation of gene transcription or mRNA translation can control individual pathways. Allosteric control of the enzyme catalyzing the first committed step of a pathway is another mechanism used by cells to regulate biosynthetic pathways. Regulatory effects resulting from interactions between pathways are less well understood, despite the fact that many metabolites are present in multiple pathways in the cell.The expanding network of defined pathways that affect the biosynthesis of thiamine provides an attractive model system to investigate metabolic integration. As depicted in Fig. 1, the biosynthetic pathway for the hydroxymethylpyrimidine (HMP) moiety of thiamine pyrophosphate (TPP) branches off from that for purine mononucleotides at the metabolite aminoimidazole ribotide (AIR). Relevant to the work presented here is a subsequent purine intermediate, aminoimidazole carboxamide ribotide (AICAR), that is also a by-product of the biosynthesis of histidine.The distribution of AIR at the purine-HMP branch point must account for the different level of purine mononucleotides and TPP required for growth (purines/TPP ratio, 1,000:1, based on auxotrophic requirements). The distribution of AIR between these pathways could be accomplished by kinetic parameters of the relevant enzymes; however, the first dedicated HMP biosynthetic enzyme is poorly understood, thus hampering direct tests of this possibility.While thiamine and purine mononucleotides are synthesized by pathways diverging at the metabolite AIR, the cellular pool of AICAR arises from both purine and histidine biosynthesis and is used exclusively for purine mononucleotide synthesis (28, 29). Since AICAR enters purine mononucleotide synthesis after the purine-HMP branch point, no interaction between AICAR and HMP synthesis was anticipated. Hence, it was surprising to find that mutants blocked in the utilization of AICAR (purH mutants) required both purines and thiamine to grow (16, 58). The hypothesis proposed to explain this requirement posited that inactivation of purH would result in the acc...

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The control mechanism of thiamine biosynthesis a model for the study of control of converging pathways

Cited by 37 publications

References 5 publications

Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium

Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium

Thiazole Synthase from Escherichia coli

Metabolic Flux in Both the Purine Mononucleotide and Histidine Biosynthetic Pathways Can Influence Synthesis of the Hydroxymethyl Pyrimidine Moiety of Thiamine in Salmonella enterica

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