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.
Highlights
Coli [3,4,5,6,7] where it is derived from D-erythrose-4-phosphate, deoxy-Dxylulose 5-phosphate, and glutamate (5, 8 –10) (Scheme 1)
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
Because YaaE genes were amplified by PCR (YaaD) activity in the presence of glutamine is dependent on a functional YaaE, we examined the effect of acivicin on pyridoxal 5-phosphate (PLP) synthesis under these conditions
Summary
Coli [3,4,5,6,7] where it is derived from D-erythrose-4-phosphate, deoxy-Dxylulose 5-phosphate, and glutamate (5, 8 –10) (Scheme 1). 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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