Identification of the Two Missing Bacterial Genes Involved in Thiamine Salvage: Thiamine Pyrophosphokinase and Thiamine Kinase

Melnick, Jonathan; Lis, Ewa; Park, Joo-Heon; Kinsland, Cynthia; Mori, Hirotada; Baba, Tomoya; Perkins, John B.; Schyns, Ghislain; Vassieva, Olga; Osterman, Andrei L.; Begley, Tadhg P.

doi:10.1128/jb.186.11.3660-3662.2004

Cited by 49 publications

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References 11 publications

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“…See the text for details. Question marks indicate possible unknown genes involved in the thiamine biosynthesis and salvage pathway (15,19). The organization of Bacillus thiamine biosynthesis genes is shown on the right.…”

Section: Methodsmentioning

confidence: 99%

“…Thiamine biosynthesis and salvage pathway of Bacillus subtilis. B. subtilis thi genes were previously identified by genetic, biochemical, and/or in silico evidence (1,19,25,26,29); in some cases, their original y gene designation is listed. E. coli intermediates or genes not present in B. subtilis but with similar functions are given in parentheses (1).…”

Section: Methodsmentioning

confidence: 99%

“…Two other salvage kinases, one specific for hydroxyethylthiazole and the other with a broad range of substrate specificities, including vitamin B 6 compounds and HMP, have been identified in B. subtilis and are encoded by the genes thiM and pdxK (ywdB), respectively (25). However, an ortholog to the E. coli gene thiK (ycfN) encoding thiamine kinase, a salvage enzyme that converts thiamine to TMP, has not been identified in B. subtilis (19). The B. subtilis thiamine biosynthesis pathway showing all known and proposed genes and intermediates is shown in Fig.…”

mentioning

confidence: 99%

“…B. subtilis contains another potential way to produce TPP, through a thiamine one-step salvage pathway formerly discovered in Saccharomyces cerevisiae (23). This pathway, initially thought to exist only in lower eukaryotes, is catalyzed by the product of the thiN (yloS) gene (19). Two other salvage kinases, one specific for hydroxyethylthiazole and the other with a broad range of substrate specificities, including vitamin B 6 compounds and HMP, have been identified in B. subtilis and are encoded by the genes thiM and pdxK (ywdB), respectively (25).…”

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Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis

et al. 2005

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In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this ⌬thiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the ⌬thiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis

et al. 2005

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“…The HET is converted to HET-P by HET kinase (ThiM) and the HMP is converted to HMP-PP by HMP kinase/HMP-P kinase (ThiD). In bacteria such as Escherichia coli and Serratia sp., whose TPP is synthesized from TP by TP kinase, incorporated thiamin is converted to TP by thiamin kinase (ThiK) (9). On the other hand, organisms having thiamin pyrophosphokinase synthesize TPP from external thiamin by direct pyrophosphorylation.…”

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Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon <i>Pyrobaculum calidifontis</i>

Hayashi

Nosaka

2015

J Nutr Sci Vitaminol

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Summary Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thiazole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thiamin phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphokinase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidifontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P. calidifontis. Furthermore, the results obtained by site-directed mutagenesis suggest that the Ser196 of ThiL protein plays a pivotal role in the catalytic process. Key Words thiamin phosphate kinase, thiamin pyrophosphate, thiamin synthesis, ThiL, Pyrobaculum calidifontis Thiamin, also known as vitamin B1, occurs in cells as free thiamin and as its phosphate esters, thiamin phosphate (TP), thiamin pyrophosphate (TPP) and thiamin triphosphate (1). TPP is a cofactor for many enzymes indispensable for glucose and energy metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and transketolase. Thiamin consists of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine, HMP) and 4-methyl-5-bhydroxyethylthiazole (hydroxyethylthiazole, HET). The de novo pathway of thiamin biosynthesis involves the independent formation of HMP pyrophosphate (HMP-PP) and HET phosphate (HET-P), as well as their subsequent condensation to form TP by thiamin phosphate synthase common in all organisms (2-4). In eubacteria and eukaryotes, ThiE protein and its orthologs play the role of thiamin phosphate synthase, whereas ThiN protein acts as the catalyst of the same reaction in most archaea (5, 6). Then the final product TPP is synthesized from TP in the de novo pathway (Fig. 1). Many eubacterial cells synthesize TPP by the phosphorylation of TP in the presence of ATP and Mg 21 by thiamin phosphate kinase (TP kinase, ThiL, EC 2.7.4.16) (7). In eukaryotic cells and some eubacterial cells, this final step occurs in two stages: hydrolysis ...

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Host Organisms:Bacillus subtilis

Hohman

Dijl²,

Krishnappa³

et al. 2016

Industrial Biotechnology

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Identification of the Two Missing Bacterial Genes Involved in Thiamine Salvage: Thiamine Pyrophosphokinase and Thiamine Kinase

Abstract: The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.

Cited by 49 publications

References 11 publications

Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis

Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis

Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon <i>Pyrobaculum calidifontis</i>

Host Organisms:Bacillus subtilis

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