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

Abstract

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...

Vitamin B6 biosynthesis in higher plants

“…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. Here, we demonstrate the reconstitution of vitamin B6 biosynthesis from a higher plant.…”

Defining the structural requirements for ribose 5‐phosphate‐binding and intersubunit cross‐talk of the malarial pyridoxal 5‐phosphate synthase

“…The activity of the PLP synthase system is highly regulated [7,[14][15][16][17][18][19]. Cooperativity between Pdx1 subunits is induced upon binding of the pentose substrate [18].…”

“…[5,19]. Formation of PLP was assayed using a Jasco V-550 spectrophotometer (Jasco, Groß-Umstadt, FRG) at a wavelength of 414 nm at 37°C for 30 min.…”

The role of the pyridoxine (vitamin B6) biosynthesis enzyme PDX1 in ultraviolet-B radiation responses in plants

“…In the alternative pyridoxine biosynthesis pathway, used by e.g. yeast, fungi, and plants, the general understanding is that pyridoxal-5-phosphate is synthesized from glutamine, ribose 5-phosphate (or ribulose 5-phosphate), and glyceraldehyde 3-phosphate (or dihydroxyacetone phosphate) by two proteins, PDX1 and PDX2, which function in a complex as a glutamine amidotransferase [16,17]. PDX2 possesses glutaminase activity.…”