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

Belitsky, Boris R.

doi:10.1128/jb.186.4.1191-1196.2004

Cited by 74 publications

(80 citation statements)

References 48 publications

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“…PDX1 and PDX2 have been predicted to function as a glutamine amidotransferase with PDX2 as the glutaminase domain and PDX1 as the acceptor͞synthase domain. Indeed, glutaminase activity has been demonstrated for PDX2 from a number of organisms (16)(17)(18). More recently, Burns et al (19) and our own independent studies (51) have been able to reconstitute vitamin B6 formation from intermediates of glycolysis and the pentose phosphate pathway by using the PDX1 and PDX2 homologs from the Gram-positive bacterium Bacillus subtilis.…”

mentioning

confidence: 94%

Vitamin B6 biosynthesis in higher plants

Tambasco-Studart

Titiz²,

Raschle³

et al. 2005

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

210

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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mentioning

confidence: 94%

Vitamin B6 biosynthesis in higher plants

Tambasco-Studart

Titiz²,

Raschle³

et al. 2005

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

210

260

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“…Moreover, 15 N-labeling studies in yeast had indicated that the nitrogen atom of pyridoxine is derived from the amide moiety of glutamine (9,10), whereas in the E. coli pathway it is derived from glutamate (10,21). Indeed, glutaminase activity has since been demonstrated for the PDX2 proteins of B. subtilis, Saccharomyces cerevisiae, and Plasmodium falciparum (22)(23)(24), designated YaaE (PdxT), SNO1, and PDX2, respectively. However, glutaminase activity was only observed in the presence of PDX1, which was shown to co-purify with PDX2, forming a hetero-oligomeric complex as has been observed for other glutamine amidotransferases (22,23).…”

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confidence: 99%

“…Indeed, glutaminase activity has since been demonstrated for the PDX2 proteins of B. subtilis, Saccharomyces cerevisiae, and Plasmodium falciparum (22)(23)(24), designated YaaE (PdxT), SNO1, and PDX2, respectively. However, glutaminase activity was only observed in the presence of PDX1, which was shown to co-purify with PDX2, forming a hetero-oligomeric complex as has been observed for other glutamine amidotransferases (22,23). In general, glutamine amidotransferases consist of two domains, a glutaminase domain, which hydrolyzes glutamine to glutamate and ammonia, and a synthase domain that accepts and utilizes the ammonia from the glutaminase domain (19).…”

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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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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“…Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B 6 and is essential for numerous enzyme reactions, including transamination, decarboxylation, racemization, and elimination in amino acid metabolism, biosynthesis of antibiotics, and DNA biosynthesis (Eliot and Kirsch, 2004;Percudani and Peracchi, 2003). As de novo PLP biosynthesis pathways exist in bacteria, fungi, protozoa, and plants, but are missing in mammals, PLP biosynthesis could be a target for developing antibiotics (Belitsky, 2004;Dong et al, 2004;Osmani et al, 1999). There are two mutually exclusive de novo pathways (deoxyxylulose 5′-phosphate (DXP)-dependent and DXP-independent) (Fitzpatrick et al, 2007;Tambasco-Studart et al, 2005).…”

Section: Introductionmentioning

confidence: 99%