Biosynthesis of Vitamin B<sub>6</sub> in Yeast:  Incorporation Pattern of Glucose

Gupta, Ram N.; Hemscheidt, Thomas; Sayer, Brian G.; Spenser, Ian D.

doi:10.1021/ja0113201

Cited by 28 publications

(38 citation statements)

References 17 publications

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“…Identification of YaaD Substrates-Based on labeling (12,14,25) and genetic studies in yeast (26), potential substrates of YaaD were considered and tested for their ability to form vitamin B6 in the presence of YaaE and glutamine in 50 mM Tris-Cl, pH 8.0. Employing the pentulose sugar ribulose 5-phosphate and either of the triose sugars glyceraldehyde 3-phosphate or dihydroxyacetone phosphate, enzymatic activity could be observed by the appearance of an absorbance maximum at 414 nm.…”

Section: Resultsmentioning

confidence: 99%

“…It was, therefore, postulated that PDX1 is likely to be the synthase subunit of the glutamine amidotransferase hetero-oligomeric complex (22)(23)(24), but the nature of the substrates in the synthase reaction remained unknown. Nonetheless, extensive labeling studies in yeast had established that the origin of the C5-chain C-2Ј,2,3,4,4Ј of pyridoxamine is derived from an intact glucose-derived pentulose and/or pentose intermediate rather than deoxy-D-xylulose s-phosphate as in E. coli (25), and more recently a genetic study in yeast indicated that the five-carbon sugar might be ribulose 5-phosphate (26), but the identity of the remaining triose sugar remained elusive (12). While this report was in preparation, enzymatic synthesis of vitamin B6 was demonstrated (27) using either of the C5 sugars, ribose 5-phosphate or ribulose 5-phosphate, and either of the C3 sugars, glyceraldehyde 3-phosphate or dihydroxyacetone phosphate, in the presence of glutamine and the PDX1 and PDX2 homologs from B. subtilis (YaaD and YaaE, respectively), but sufficient proof of the identity of the reaction product was not provided.…”

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confidence: 99%

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On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

Raschle

Amrhein

Fitzpatrick

2005

Journal of Biological Chemistry

118

163

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Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.In humans vitamin B6 is required for the maintenance of health. From a biochemical point of view the vitamin is well recognized in its active form as pyridoxal 5Ј-phosphate (PLP), 2 one of nature's most versatile cofactors and required for many diverse enzymes ranging from amino acid metabolism to the biosynthesis of antibiotic compounds. Furthermore, very recently, the B6 vitamers have been shown to be potent antioxidants equivalent to vitamins C and E, effectively quenching the reactive oxygen species singlet oxygen and superoxide anion in vitro (1, 2). Vitamin B6 is synthesized in bacteria, fungi, and plants; yet, despite its essential role, its biosynthesis has been studied at the enzymatic level essentially only in the Gram-negative bacterium Escherichia coli (3-7) where it is derived from D-erythrose-4-phosphate, deoxy-Dxylulose 5-phosphate, and glutamate (5, 8 -10) (Scheme 1). The biosynthesis of the vitamin has recently come back into scrutiny, because it has become clear from studies in fungi that a pathway different from that in E. coli exists (1,(11)(12)(13)(14).Two of the key genes involved in this novel pathway have been identified (PDX1 and PDX2) (1,(15)(16)(17), neither of which show similarity to any of the E. coli genes (1, 15, 16). At the outset, amino acid sequence alignment studies indicated t...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

Raschle

Amrhein

Fitzpatrick

2005

Journal of Biological Chemistry

118

163

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“…In yeast, the nonmevalonate pathway of isoprenoid biosynthesis does not exist (46). Moreover, the extensive studies of Spenser and colleagues (13,14) indicate that in yeast, vitamin B6 biosynthesis proceeds by a route distinct from that in E. coli in that a pentose, which is not DXP, and a triose are the sugar precursors for formation of the vitamin in this organism. In the bacterial model (B. subtilis), it has recently been established by Burns et al (19), and indeed by our own independent studies (51), that YaaD (the PDX1 homolog) can accept either ribose 5-phosphate or ribulose 5-phosphate as the pentose sugar and either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate as the triose sugar to form vitamin B6 in the presence of YaaE (the PDX2 homolog) and glutamine.…”

Section: Vitamin B6 Biosynthesis In Plants Occurs Independent Of Deoxmentioning

confidence: 99%

“…Recently, it has become clear from genetic and labeling studies in fungi (2,(11)(12)(13)(14) that a pathway alternative to the one in E. coli that does not involve DXP exists for vitamin B6 biosynthesis. Only two genes (PDX1 and PDX2), which show no homology to any of the E. coli genes, appear to be involved in this alternative pathway (2,15).…”

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confidence: 99%

Vitamin B6 biosynthesis in higher plants

Tambasco-Studart

Titiz²,

Raschle³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

212

260

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Vitamin B6 is an essential metabolite in all organisms. It can act as a coenzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have a de novo biosynthetic pathway for vitamin B6, but animals must obtain it from dietary sources. In Escherichia coli, it is known that the vitamin is derived from deoxyxylulose 5-phosphate (an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis) and 4-phosphohydroxy-L-threonine. It has been assumed that vitamin B6 is synthesized in the same way in plants, but this hypothesis has never been experimentally proven. Here, we show that, in plants, synthesis of the vitamin takes an entirely different route, which does not involve deoxyxylulose 5-phosphate but instead utilizes intermediates from the pentose phosphate pathway, i.e., ribose 5-phosphate or ribulose 5-phosphate, and from glycolysis, i.e., dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. The revelation is based on the recent discovery that, in bacteria and fungi, a novel pathway is in place that involves two genes (PDX1 and PDX2), neither of which is homologous to any of those involved in the previously doctrined E. coli pathway. We demonstrate that Arabidopsis thaliana has two functional homologs of PDX1 and a single homolog of PDX2. Furthermore, and contrary to what was inferred previously, we show that the pathway appears to be cytosolic and is not localized to the plastid. Last, we report that the single PDX2 homolog is essential for plant viability.Arabidopsis ͉ isoprenoid ͉ pyridoxine ͉ deoxyxylulose 5-phosphate V itamin B6 is well renowned in the medical field for being involved in more bodily functions than any other single nutrient. The vitamin exists in various forms, i.e., pyridoxal, pyridoxine, pyridoxamine, and their phosphorylated derivatives. As pyridoxal 5Ј-phosphate, it is an essential cofactor for numerous metabolic enzymes including amino acid metabolism and antibiotic biosynthesis. Most interestingly, it has recently been found that the vitamin is a potent antioxidant with a particular ability to quench reactive oxygen species such as superoxide and singlet oxygen (1, 2). The de novo biosynthesis of vitamin B6 takes place in microorganisms and plants, but this ability has been lost in animals, making it essential in the human diet.Despite the vitamin's obvious physiological and pharmaceutical importance, its biosynthesis has predominantly been studied in the Gram-negative bacterium, Escherichia coli, where it is derived from deoxyxylulose 5-phosphate (DXP, an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis) and 4-phosphohydroxy-L-threonine (refs. 3-7 and Scheme 1). Astonishingly, although plants are a major source of vitamin B6 in the human diet, our understanding of the pathway therein is very limited and has been assumed to be derived the same way as in E. coli. There is one preliminary study that reports on its biosynthesis in spinach (8), whereas other, more in-depth studies have addressed the...

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“…In Saccharomyces cerevisiae, a glucose-derived five-carboncontaining compound and a triose are utilized for PLP synthesis (13,48). No other components of the PDX1/PDX2 pathway, other than glutamine, are known, and it is unclear whether all steps of the PDX1/PDX2 pathway are common in different organisms.…”

Section: Figmentioning

confidence: 99%

Physical and Enzymological Interaction of Bacillus subtilis Proteins Required for De Novo Pyridoxal 5′-Phosphate Biosynthesis

Belitsky

2004

J Bacteriol

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Bacillus subtilis synthesizes pyridoxal 5-phosphate, the active form of vitamin B 6 , by a poorly characterized pathway involving the yaaD and yaaE genes. The pdxS (yaaD) mutant was confirmed to be a strict B 6 auxotroph, but the pdxT (yaaE) mutant turned out to be a conditional auxotroph depending on the availability of ammonium in the growth medium. The PdxS and PdxT proteins copurified during affinity chromatography and apparently form a complex that has glutaminase activity. PdxS and PdxT appear to encode the synthase and glutaminase subunits, respectively, of a glutamine amidotransferase of as-yet-unknown specificity essential for B 6 biosynthesis.

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Biosynthesis of Vitamin B₆ in Yeast: Incorporation Pattern of Glucose

Cited by 28 publications

References 17 publications

On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

Vitamin B6 biosynthesis in higher plants

Physical and Enzymological Interaction of Bacillus subtilis Proteins Required for De Novo Pyridoxal 5′-Phosphate Biosynthesis

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Biosynthesis of Vitamin B6 in Yeast: Incorporation Pattern of Glucose

Cited by 28 publications

References 17 publications

On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

Vitamin B6 biosynthesis in higher plants

Physical and Enzymological Interaction of Bacillus subtilis Proteins Required for De Novo Pyridoxal 5′-Phosphate Biosynthesis

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Biosynthesis of Vitamin B₆ in Yeast: Incorporation Pattern of Glucose