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Pyridoxal 5'-phosphate (the active form of vitamin B6) is a cofactor that is important for a broad number of biochemical reactions and is essential for all forms of life. Organisms that can synthesize pyridoxal 5'-phosphate use either the deoxyxylulose phosphate-dependent or -independent pathway, the latter is encoded by a two-component pyridoxal 5'-phosphate synthase. Saccharomyces cerevisiae contains three paralogs of the two-component SNZ/SNO pyridoxal 5'-phosphate synthase. Past work identified the biochemical activity of Snz1p, Sno1p and provided in vivo data that SNZ1 was involved in pyridoxal 5'-phosphate biosynthesis. Snz2p and Snz3p were considered redundant isozymes and no growth condition requiring their activity was reported. Genetic data herein showed that either SNZ2 or SNZ3 are required for efficient thiamine biosynthesis in Saccharomyces cerevisiae. Further, SNZ2 or SNZ3 alone could satisfy the cellular requirement for pyridoxal 5'-phosphate (and thiamine), while SNZ1 was sufficient for pyridoxal 5'-phosphate synthesis only if thiamine was provided. qRT-PCR analysis determined that SNZ2,3 are repressed ten-fold by the presence thiamine. In total, the data were consistent with a requirement for PLP in thiamine synthesis, perhaps in the Thi5p enzyme, that could only be satisfied by SNZ2 or SNZ3. Additional data showed that Snz3p is a pyridoxal 5'-phosphate synthase in vitro and is sufficient to satisfy the pyridoxal 5'-phosphate requirement in Salmonella enterica when the medium has excess ammonia. [ABSTRACT FROM AUTHOR]

Abstract: The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis. Structured summary: MINT-7147859: PDX1 (uniprotkb:P16451) and PDX1 (uniprotkb:P16451) bind (MI:0407) by cosedimentation in solution (MI:0028) MINT-7147899: PDX1 (uniprotkb:P37528) and PDX1 (uniprotkb:P37528) bind (MI:0407) by cosedimentation in solution (MI:0028) [Copyright &y& Elsevier]

Abstract: Transgenic expression of vitamin biosynthetic genes has been investigated for over-production of these dietary supplements in microorganisms and plants. In plants, successful efforts have been reported with Vitamins A, C, E and B-9, however information is lacking for other vitamins. Vitamin B-6 is an essential cofactor for numerous enzymatic reactions, and has also been shown to be a potent antioxidant involved in protecting phytopathogenic Cercospora fungi from their own toxin, cercosporin. In this report, we transformed and expressed two Vitamin B-6 biosynthetic genes (PDX1 and PDX2) isolated from Cercospora nicotianae in cercosporin-sensitive organisms, tobacco and the fungal species Aspergillus flavus and Neurospora crassa. Our goal was to determine if Vitamin B-6 levels could be increased by constitutive expression of these genes, and if over-production confers resistance to oxidative stresses induced by cercosporin and salinity stress. Elevated Vitamin B-6 levels were observed in one tobacco line. For other lines evaluated in this work, expression of PDX1 and PDX2 in transgenic organisms did not result in a significant increase in Vitamin B-6 content over controls. Analysis of gene expression in tobacco indicated that the lack of elevated B-6 content was not due to lack of enzymatic activity, but to down-regulation of the endogenous tobacco genes compounded with limited transgene expression. The single line with elevated B-6 levels had higher expression of both the PDX1 and PDX2 transgenes compared to the other lines, and the observed increase on Vitamin B-6 was correlated with higher enzyme activity. Consistent with our inability to elevate cellular B-6 levels, only small changes were observed in the response to either cercosporin or high salt, and most transgenic individuals were as susceptible as controls. Compared to tobacco lines transformed to express either PDX1 or PDX2 alone, half of the transgenic tobacco lines expressing both genes were impaired in seed germination and initial growth. However no correlation was observed between the observed phenotype and Vitamin B-6 levels in seeds. This is the first report on genetic engineering to manipulate the Vitamin B-6 pathway in plants. Our results suggest that genetic manipulation of the Vitamin B-6 biosynthetic pathway is possible but is limited by regulation of endogenous genes. [Copyright &y& Elsevier]

Abstract: Vitamin B6, an essential cofactor in enzymatic reactions, has only recently been linked to cellular oxidative stress. We investigated the role of this vitamin as an antioxidant in oxidative responses linked to plant defense. B6 vitamers effectively quenched superoxide and had antioxidant activity when assayed in vitro. The de novo B6 biosynthetic genes (PDX1 and PDX2) were identified in Nicotiana tabacum cv. ‘Burley 21’ and their transcript abundance was assayed during defense responses. PDX1 and PDX2 transcript levels decreased following inoculation with the incompatible pathogen Pseudomonas syringae pv. phaseolicola and transiently increased in response to salicylic acid and methyl jasmonate. Excess vitamin B6 in tobacco leaves interfered with the development of a hypersensitive response caused by P. syringae pv. phaseolicola and increased disease severity caused by the compatible bacterium P. syringae pv. tabaci. Our findings indicate that during plant defense responses, vitamin B6 functions and its synthesis is regulated in a manner consistent with this vitamin''s activity as an antioxidant and modulator of active oxygen species in vivo. [Copyright &y& Elsevier]

The pathway for de novo vitamin B6 biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2, have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae. Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B6 pathway encodes a protein responsible for synthesis of the pyridoxine ring. [Copyright &y& Elsevier]