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A number of organic synthesis involve threonine aldolase (TA), a pyridoxal phosphate (PLP)-dependent enzyme. Although the addition of exogenous PLP is necessary for the reactions, it increases the cost and complicates the purification of the product. This work constructed a PLP self-sufficient biocatalysis system for TA, which included an improvement of the intracellular PLP level and co-immobilization of TA with PLP. Engineered strain BL-ST was constructed by introducing PLP synthase PdxS/T to Escherichia coli BL21(ED3). The intracellular PLP concentration of the strain increased approximately fivefold to 48.5 μmol/g DCW . l-TA, from Bacillus nealsonii (BnLTA), was co-expressed in the strain BL-ST with PdxS/T, resulting in the engineered strain BL-BnLTA-ST. Compared with the control strain BL-BnLTA (254.1 U/L), the enzyme activity of the strain BL-BnLTA-ST reached 1518.4 U/L without the addition of exogenous PLP. An efficient co-immobilization system was then designed. The epoxy resin LX-1000HFA wrapped by polyethyleneimine (PEI) acted as a carrier to immobilize the crude enzyme solution of the strain BL-BnLTA-ST mixed with an extra 100 μM of exogenous PLP, resulting in the catalyst HFA PEI -BnLTA-STPLP 100. HFA PEI -BnLTA-STPLP 100 exhibited a half-life of approximately 450 h, and the application of the catalyst in the continuous biosynthesis of 3-[4-(methylsulfonyl) phenyl] serine had more than 180 batch reactions (>60% conv ) without the extra addition of exogenous PLP. The excellent compatibility and stability of the system were further confirmed by other TAs. This work introduced a PLP self-sufficient biocatalysis system that can reduce the cost of PLP and contribute to the industrial application of TA. In addition, the system may also be applied in other PLP-dependent enzymes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pyridoxal 5'-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA-PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET-TPL-pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21-TPLST-Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 μM., (Copyright © 2020. Published by Elsevier B.V.)

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host's metabolic processes or even absent in the host. In this context, the essential presence of vitamin B 6 biosynthesis enzymes in Plasmodium , the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium viva x PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1-Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.

Pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK) cooperate to produce pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PDXK phosphorylates pyridoxine, pyridoxamine, and pyridoxal by producing PNP, PMP, and PLP, whereas PNPO oxidizes PNP, PMP, into PLP. We previously demonstrated that PDXK depletion in Drosophila and human cells impacts on glucose metabolism and DNA integrity. Here we characterized sgll, the Drosophila ortholog of PNPO gene, showing that its silencing by RNA interference elicits chromosome aberrations (CABs) in brains and induces diabetic hallmarks such as hyperglycemia and small body size. We showed that in sgll RNAi neuroblasts CABs are largely produced by the genotoxic effect of the advanced glycation end products triggered by high glucose. As in sgll RNAi cells, part of PLP is still produced by PDXK activity, these data suggest that PLP dosage need to be tightly regulated to guarantee glucose homeostasis and DNA integrity., (© 2019 Wiley Periodicals, Inc.)

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP) in Escherichia coli Surprisingly, incubation of the ∆ pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

In eukaryotes, pyridoxal kinase (PDXK) acts in vitamin B 6 salvage pathway to produce pyridoxal 5'-phosphate (PLP), the active form of the vitamin, which is implicated in numerous crucial metabolic reactions. In Drosophila, mutations in the dPdxk gene cause chromosome aberrations (CABs) and increase glucose content in larval hemolymph. Both phenotypes are rescued by the expression of the wild type human PDXK counterpart. Here we expressed, in dPdxk 1 mutant flies, four PDXK human variants: three (D87H, V128I and H246Q) listed in databases, and one (A243G) found in a genetic screening in patients with diabetes. Differently from human wild type PDXK, none of the variants was able to completely rescue CABs and glucose content elicited by dPdxk 1 mutation. Biochemical analysis of D87H, V128I, H246Q and A243G proteins revealed reduced catalytic activity and/or reduced affinity for PLP precursors which justify this behavior. Although these variants are rare in population and carried in heterozygous condition, our findings suggest that in certain metabolic contexts and diseases in which PLP levels are reduced, the presence of these PDXK variants could threaten genome integrity and increase cancer risk.

Pyridoxal 5'-phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy-xylulose-5-phosphate (DXP)-independent pathway, which is present in the Gram-positive model bacterium Bacillus subtilis and the longer DXP-dependent pathway, which has been intensively studied in the Gram-negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so-called 'underground metabolism', which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non-native DXP-dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non-native enzymes catalysing the last steps of the DXP-dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non-native vitamin B6 pathway. The introduction of non-native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the Actinobacillus pleuropneumoniae S-8 genome sequence revealed the presence of pdxS and pdxT genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in A. pleuropneumoniae pathogenicity. Our data demonstrated that A. pleuropneumoniae could synthesize PLP by PdxS and PdxT enzymes. Disruption of the pdxS and pdxT genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for A. pleuropneumoniae growth and viability. Additionally, the pdxS and pdxT deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the pdxS and pdxT deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of A. pleuropneumoniae. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.

Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of Mycobacterium tuberculosis (Mtb). PLP functions as a cofactor for 58 putative PLP-binding proteins encoded by the Mtb genome and could also act as a potential antioxidant. De novo biosynthesis of PLP in Mtb takes place through the 'deoxyxylulose 5'-phosphate (DXP)-independent' pathway, whereas PdxH enzymes, possessing pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPOx) activity, are involved in the PLP salvage pathway. In this study, we demonstrate that the annotated PdxH enzymes from various mycobacterial species are bona fide members of the classical PNPOx enzyme family, capable of producing PLP using both pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) substrates., (© 2016 Federation of European Biochemical Societies.)

Although the routes of de novo pyridoxal 5'-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis.

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5'-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for "green" manufacturing processes.

The origin and evolution of modern biochemistry remain a mystery despite advances in evolutionary bioinformatics. Here, we use a structural census in nearly 1,000 genomes and a molecular clock of folds to define a timeline of appearance of protein families linked to single-domain enzymes. The timeline sorts out enzymatic recruitment, validates patterns in metabolic history, and reveals that the most ancient reaction of aerobic metabolism involved the synthesis of pyridoxal 5'-phosphate or pyridoxal and appeared 2.9 Gyr ago. The oxygen source for this primordial reaction was probably Mn catalase, which appeared at the same time and could have generated oxygen as a side product of hydrogen peroxide detoxification. Finally, evolutionary analysis of transferred groups and metabolite fragments revealed that oxidized sulfur did not participate in metabolism until the rise of oxygen. The evolutionary patterns we uncover in molecules and chemistries provide strong support for the coevolution of biochemistry and geochemistry., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B(6) (pyridoxal 5'-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alternative positioning of an α helix distinguishes an open conformation from a closed state when the enzyme binds substrate. The pentose substrate is covalently attached through its C1 and forms a Schiff base with Lys84. Ammonia transfer between Pdx2 glutaminase and Pdx1 active sites is regulated by a transient tunnel. The mutagenesis analysis allows defining the requirement for conservation of critical methionines, whereas there is also plasticity in ammonia tunnel construction as seen from comparison across different species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Vitamin B(6) is an essential cofactor that participates in a large number of biochemical reactions. Pyridoxal phosphate is biosynthesized de novo by two different pathways (the DXP dependent pathway and the R5P pathway) and can also be salvaged from the environment. It is one of the few cofactors whose catabolic pathway has been comprehensively characterized. It is also known to function as a singlet oxygen scavenger and has protective effects against oxidative stress in fungi. Enzymes utilizing vitamin B(6) are important targets for therapeutic agents. This review provides a concise overview of the mechanistic enzymology of vitamin B(6) biosynthesis and catabolism. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology., (Copyright © 2011. Published by Elsevier B.V.)

The pdxR (cg0897) gene of Corynebacterium glutamicum ATCC 13032 encodes a regulatory protein belonging to the MocR subfamily of GntR-type transcription regulators and consisting of an amino-terminal winged helix-turn-helix DNA-binding domain and a carboxy-terminal aminotransferase-like domain. A defined deletion in the pdxR gene resulted in the decreased expression of the divergently orientated pdxST genes coding for the subunits of pyridoxal 5'-phosphate synthase. The pdxST mutant C. glutamicum NJ0898 and the pdxR mutant C. glutamicum AMH17 showed vitamin B(6) auxotrophy that was restored by supplementing the growth medium with either pyridoxal, pyridoxal 5'-phosphate or pyridoxamine. The genetic organization of the 89 bp pdxR-pdxST intergenic region was elucidated by mapping the 5' ends of the respective transcripts, followed by detection of typical promoter sequences. Bioinformatic pattern searches and comparative genomics revealed three DNA motifs with the consensus sequence AAAGTGGW(-/T)CTA, overlapping the deduced promoter sequences and serving as candidate DNA-binding sites for PdxR. DNA band shift assays with the purified PdxR protein demonstrated the specific binding of the transcription regulator to double-stranded 40-mer sequences containing the detected motifs, thereby confirming the direct regulatory role of PdxR in activating the expression of the pdxST genes.

Bacterial genomes encode hundreds to thousands of enzymes, most of which are specialized for particular functions. However, most enzymes have inefficient promiscuous activities, as well, that generally serve no purpose. Promiscuous reactions can be patched together to form multistep metabolic pathways. Mutations that increase expression or activity of enzymes in such serendipitous pathways can elevate flux through the pathway to a physiologically significant level. In this study, we describe the discovery of three serendipitous pathways that allow synthesis of pyridoxal-5'-phosphate (PLP) in a strain of E. coli that lacks 4-phosphoerythronate (4PE) dehydrogenase (PdxB) when one of seven different genes is overexpressed. We have characterized one of these pathways in detail. This pathway diverts material from serine biosynthesis and generates an intermediate in the normal PLP synthesis pathway downstream of the block caused by lack of PdxB. Steps in the pathway are catalyzed by a protein of unknown function, a broad-specificity enzyme whose physiological role is unknown, and a promiscuous activity of an enzyme that normally serves another function. One step in the pathway may be non-enzymatic.

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-d-erythronate (4PE) to 2-oxo-3-hydroxy-4-phosphobutanoate (OHPB) with concomitant reduction of NAD(+) to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD(+) does not reoxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-keto acids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (k(cat)/K(m) values ~1 × 10(4) M⁻¹s⁻¹). The kinetic parameters for the substrate 4PE include a k(cat) of 1.4 s⁻¹, a K(m) of 2.9 μM, and a k(cat)/K(m) of 6.7 × 10(6) M⁻¹s⁻¹. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that d-2-HGA, but not l-2-HGA, is a competitive inhibitor vs 4PE and a noncompetitive inhibitor vs α-ketoglutarate.

Most organisms synthesise the B(6) vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I(320), and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Vitamin B6 comprises a group of compounds that are involved in a surprisingly high diversity of biochemical reactions. Actually, most of these reactions are co-catalyzed by a single B6 vitamer, pyridoxal 5'-phosphate, making it a crucial and versatile co-factor in many metabolic processes in the cell. In addition, it has been demonstrated in recent years that vitamin B6 has a second important function by being an effective antioxidant. Because of these two characteristics the vitamin is an interesting compound to study in plants. This review provides a brief overview and update on such important aspects like vitamin B6-dependent enzymes and known biosynthetic pathways in plants, phenotypes of plant mutants affected in vitamin B6 biosynthesis, and the potential benefits of modifying vitamin B6 content in plants., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Vitamin B(6) is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5'-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A glutamine amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and glutamine.

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate.

As salt stress imposes a major environmental threat to agriculture, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any transgenic strategy. Salt Overly Sensitive (SOS) genes (SOS1-SOS3) were isolated through positional cloning. Since sos mutants are hypersensitive to salt, their characterization resulted in the discovery of a novel pathway, which has helped in our understanding the mechanism of salt-stress tolerance in plants. Genetic analysis confirmed that SOS1-SOS3 function in a common pathway of salt tolerance. This pathway also emphasizes the significance of Ca2+ signal in reinstating cellular ion homeostasis. SOS3, a Ca2+ sensor, transduces the signal downstream after activating and interacting with SOS2 protein kinase. This SOS3-SOS2 complex activates the Na+/H+ antiporter activity of SOS1 thereby reestablish cellular ion homeostasis. Recently, SOS4 and SOS5 have also been characterized. SOS4 encodes a pyridoxal (PL) kinase that is involved in the biosynthesis of pyridoxal-5-phosphate (PLP), an active form of vitamin B6. SOS5 has been shown to be a putative cell surface adhesion protein that is required for normal cell expansion. Under salt stress, the normal growth and expansion of a plant cell becomes even more important and SOS5 helps in the maintenance of cell wall integrity and architecture. In this review we focus on the recent advances in salt stress and SOS signaling pathway. A broad coverage of the discovery of SOS mutants, structural aspect of these genes and the latest developments in the field of SOS1-SOS5 has been described.

Two routes for the de novo biosynthesis of pyridoxal-5'-phosphate (PLP) have been discovered and reconstituted in vitro. The most common pathway that organisms use is dependent upon the activity of just two enzymes, known as Pdx1 (YaaD) and Pdx2 (YaaE) in bacteria. Pdx2 has been shown to have glutaminase activity and most likely channels ammonia to the active site of the PLP synthase subunit, Pdx1, where ribose-5-phosphate (R5P), glyceraldehyde-3-phosphate (G3P), and ammonia are condensed in a complex series of reactions. In this report we investigated the early steps in the mechanism of PLP formation. Under pre-steady-state conditions, a chromophoric intermediate (I320) is observed that accumulates upon addition of only two of the substrates, R5P and glutamine. The intermediate is covalently bound to the protein. We synthesized C5 monodeuterio (pro-R, pro-S) and dideuterio R5P and showed that there is a primary kinetic isotope effect on the formation of this intermediate using the pro-R but not the pro-S labeled isomer. Furthermore, it was shown that the phosphate unit of R5P is eliminated rather than hydrolyzed in route to intermediate formation and also that there is an observed C5-deuterium kinetic isotope effect on this elimination step. Interestingly, it was observed that the formation of the intermediate could be triggered in the absence of Pdx2 by the addition of high concentrations of NH4Cl to a preincubated solution of Pdx1 and R5P. The formation of I320 was also monitored using high-resolution electrospray ionization Fourier transform mass spectrometry and revealed a species of mass 34,304 Da (Pdx1 + 95 Da). These results allow us to narrow the mechanistic possibilities for the complex series of reactions involved in PLP formation.

In the present study, we investigated ischemia-induced changes of pyridoxal 5'-phosphate synthesizing enzyme and degrading enzyme and neuroprotective effects and roles of pyridoxal 5'-phosphate against ischemic damage in the gerbil hippocampal CA1 region. Pyridoxal 5'-phosphate oxidase and pyridoxal phosphate phosphatase immunoreactivities were changed in neurons up to 2 days after ischemia, while 4 days after ischemia their immunoreactivities were expressed in astrocytes. Pyridoxal 5'-phosphate oxidase immunoreactivity and its protein level were highest 12 h after ischemia, while those in pyridoxal phosphate phosphatase were highest 2 days after ischemia. Total activities of these enzymes were changed after ischemia, but specific activities of the enzymes were not altered. Treatment with pyridoxal 5'-phosphate into brains (4 microg/5 microl, i.c.v.) at 30 min before transient ischemia protected about 80% of CA1 pyramidal cells 4 days after ischemia and induced elevation of glutamic acid decarboxylase 67 immunoreactivity in the CA1 region. However, pyridoxal 5'-phosphate treatment into ischemic brains decreased GABA transaminase immunoreactivity in the CA1 region after ischemia. These results indicate that pyridoxal 5'-phosphate may be associated with the inhibitory discharge of GABA in the hippocampal CA1 neurons, and the increased level of GABA may protect hippocampal CA1 pyramidal cells from ischemic damage.

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.

Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.

Vitamin B(6) (VB6) functions as a cofactor of many diverse enzymes in amino acid metabolism. Three metabolic pathways for pyridoxal 5'-phosphate (PLP; the active form of VB6) are known: the de novo pathway, the salvage pathway, and the fungal type pathway. Most unicellular organisms and plants biosynthesize VB6 using one or two of these three biosynthetic pathways. However, animals such as insects and mammals do not possess any of the pathways and, thus, need to intake VB6 in their diet to survive. It is conceivable that breakdowns of these pathways occurred in the evolutionary lineages of insects and mammals, and one of the major reasons for this would be the loss of pertinent genes. We studied the evolution of VB6 biosynthesis from the view of the gain and loss of 10 pertinent genes in 122 species whose genome sequences were completely determined. The results revealed that each gene in the pathways was lost more than once in the entire evolutionary lineages of the 122 species. We also found the following three points regarding the evolution of PLP biosynthesis: (1) the breakdown of the PLP biosynthetic pathways occurred independently at least three times in animal lineages, (2) the de novo pathway was formed by the generation of pdxB in gamma-proteobacteria, and (3) the order of the gene loss in VB6 metabolism was conserved among different evolutionary lineages. These results suggest that the evolution of VB6 metabolism was subject to gains and frequent losses of related genes in the 122 species examined. This dynamic nature of the evolutionary changes must have been responsible for the breakdowns of the pathways, resulting in profound differentiation of heterotrophy among the species.

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.

Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined. The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason. The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP. The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis.

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.

The structure of YaaE from Bacillus subtilis was determined at 2.5-A resolution. YaaE is a member of the triad glutamine aminotransferase family and functions in a recently identified alternate pathway for the biosynthesis of vitamin B(6). Proposed active residues include conserved Cys-79, His-170, and Glu-172. YaaE shows similarity to HisH, a glutaminase involved in histidine biosynthesis. YaaD associates with YaaE. A homology model of this protein was constructed. YaaD is predicted to be a (beta/alpha)(8) barrel on the basis of sequence comparisons. The predicted active site includes highly conserved residues 211-216 and 233-235. Finally, a homology model of a putative YaaD-YaaE complex was prepared using the structure of HisH-F as a model. This model predicts that the ammonia molecule generated by YaaE is channeled through the center of the YaaD barrel to the putative YaaD active site.

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.

Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.

Pyridoxine 5'-phosphate (PNP) synthase is the last enzyme in the de novo biosynthesis of vitamin B(6) catalyzing the complicated ring-closure reaction between 1-deoxy-D-xylulose-5-phosphate and 1-amino-acetone-3-phosphate. Here we present the crystal structures of four PNP synthase complexes with substrates and substrate analogs. While the overall fold of the enzyme is conserved in all complexes, characteristic readjustments were observed in the active site. The complementary structural information allowed us to postulate a detailed reaction mechanism. The observed binding mode of substrates indicates how the first reaction intermediate, the Schiff-base conjugate, is formed. The most important mechanistic features are the presence of two phosphate-binding sites with distinct affinities and the existence of a water relay system for the release of reaction water molecules. Furthermore, the complexes provide the basis to rationalize the open-closed transition of a flexible loop located on the C-terminal side of the TIM-barrel. Binding of both substrate molecules to the active site seems to be a prerequisite to trigger this transition. Highly conserved mechanistically important residues in the PNP synthase family imply a similar active site organization and reaction mechanism for all family members. Due to the exclusive presence of PNP synthase in a subset of eubacteria, including several well-known pathogens, and due to its outstanding physiological importance for these organisms, the enzyme appears to be a promising novel target for antibacterial drug design.

Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis. We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene. sos4 mutant plants are hypersensitive to Na(+), K(+), and Li(+) ions. Under NaCl stress, sos4 plants accumulate more Na(+) and retain less K(+) compared with wild-type plants. SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses. Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels. Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na(+) and K(+) homeostasis in plants. We propose that pyridoxal-5-phosphate regulates Na(+) and K(+) homeostasis by modulating the activities of ion transporters.

Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.

Vitamin B6 in its active form pyridoxal phosphate is an essential coenzyme of many diverse enzymes. Biochemistry, enzymology and genetics of de novo vitamin B6 biosynthesis have been primarily investigated in Escherichia coli. Database searches revealed that the key enzymes involved in ring closure of the aromatic pyridoxin ring (PdxA; PdxJ) are present mainly in genomes of bacteria constituting the gamma subdivision of proteobacteria. The distribution of DXS, a transketolase-like enzyme involved in vitamin B6 biosynthesis as well as in thiamine and isoprenoid biosynthesis and the distribution of vitamin B6 modifying enzymes (PdxH: oxidase; PdxK: kinase) was also analyzed. These enzymes are also present in the genomes of animals. Two recent papers (Ehrenshaft et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 9374-9378; Osmani et al., 1999, J. Biol. Chem. 274: 23565-23569) show the involvement of an extremely conserved protein (a member of the UPF0019 or SNZ family) found in all three domains of life (bacteria, archaea, eukarya) in an alternative vitamin B6 biosynthesis pathway. Members of this family were previously identified as a stationary phase inducible protein in yeast, as an ethylene responsible protein in plants and in a marine sponge, as a singlet oxygen resistance protein in Cercospora nicotianae and as a cumene hydroperoxide and H2O2 inducible protein in Bacillus subtilis. In yeast, the SNZ protein interacts with another protein called SNO which also represents a member of a highly conserved protein family (called UPF0030 or SNO family). Phylogenetic trees for the DXS, PdxA, PdxJ, PdxH, PdxK, SNZ and SNO protein families are presented and possible implications of the two different vitamin B6 biosynthesis pathways in cellular metabolism are discussed. A radically different view of bacterial evolution (Gupta, 2000, Crit. Rev. Microbiol. 26: 111-131) which proposes a linear rather than a treelike evolutionary relationship between procaryotic species indicates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. This proposal might help to explain why the PdxA/PdxJ pathway is largely restricted to this subdivision.

The enzyme PdxJ catalyzes the condensation of 1-deoxy-D-xylulose-5-phosphate (DXP) and 1-amino-3-oxo-4-(phosphohydroxy)propan-2-one to form pyridoxine 5'-phosphate (PNP). The protein from Escherichia coli has been crystallized in several forms under different conditions. The best diffracting crystals were obtained by a combination of the hanging-drop vapour-diffusion and microseeding techniques. Using an in-house image plate, the PdxJ crystals diffracted under cryo-conditions to 2.6 A resolution. The space group has been determined as C222(1), with unit-cell parameters a = 132.5, b = 154. 4, c = 131.4 A, corresponding to four monomers per asymmetric unit. In the search for heavy-atom derivatives, a mercury derivative has been interpreted. The 12 mercury sites located are related by 222 symmetry and, in combination with self-rotation search analyses and gel-filtration experiments, indicate the quaternary assembly of PdxJ into octamers with 422 symmetry.

Because exercise stresses metabolic pathways that depend on thiamine, riboflavin, and vitamin B-6, the requirements for these vitamins may be increased in athletes and active individuals. Theoretically, exercise could increase the need for these micronutrients in several ways: through decreased absorption of the nutrients; by increased turnover, metabolism, or loss of the nutrients; through biochemical adaptation as a result of training that increases nutrient needs; by an increase in mitochondrial enzymes that require the nutrients; or through an increased need for the nutrients for tissue maintenance and repair. Biochemical evidence of deficiencies in some of these vitamins in active individuals has been reported, but studies examining these issues are limited and equivocal. On the basis of metabolic studies, the riboflavin status of young and older women who exercise moderately (2.5-5 h/wk) appears to be poorer in periods of exercise, dieting, and dieting plus exercise than during control periods. Exercise also increases the loss of vitamin B-6 as 4-pyridoxic acid. These losses are small and concomitant decreases in blood vitamin B-6 measures have not been documented. There are no metabolic studies that have compared thiamine status in active and sedentary persons. Exercise appears to decrease nutrient status even further in active individuals with preexisting marginal vitamin intakes or marginal body stores. Thus, active individuals who restrict their energy intake or make poor dietary choices are at greatest risk for poor thiamine, riboflavin, and vitamin B-6 status.

In Escherichia coli the coenzyme pyridoxal 5'-phosphate (PLP) is synthesised de novo by a pathway that is thought to involve the condensation of 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose, catalysed by the enzymes PdxA and PdxJ, to form either pyridoxine (vitamin B6) or pyridoxine 5'-phosphate (PNP). Here we show that incubation of PdxJ with PdxA, 4-(phosphohydroxy)-L-threonine, NAD and 1-deoxy-D-xylulose-5-phosphate, but not 1-deoxy-D-xylulose, results in the formation of PNP. The PNP formed was characterised by (i) cochromatography with an authentic standard, (ii) conversion to pyridoxine by alkaline phosphatase treatment, and (iii) UV and fluorescence spectroscopy. Furthermore, when [2-(14)C]1-deoxy-D-xylulose-5-phosphate was used as a substrate, the radioactivity was incorporated into PNP. These results clarify the previously unknown role of PdxJ in the de novo PLP biosynthetic pathway. The sugar used as substrate by PdxJ is 1-deoxy-D-xylulose-5-phosphate rather than the previously assumed 1-deoxy-D-xylulose. The first vitamin B6 vitamer synthesised is PNP, and not pyridoxine.

We show that epd (gapB) mutants lacking an erythrose 4-phosphate (E4P) dehydrogenase are impaired for growth on some media and contain less pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) than their epd+ parent. In contrast to a previous report, we found that gapA epd double mutants lacking the glyceraldehyde 3-phosphate and E4P dehydrogenases are auxotrophic for pyridoxine. These results implicate the GapA and Epd dehydrogenases in de novo PLP and PMP coenzyme biosynthesis.

pdrK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5'-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY+ overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette into pdxY and crossed the resulting pdxY::omegaKan(r) mutation into the bacterial chromosome of a pdrB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts of pdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6 vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show that pdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNA(Tyr) synthetase) in a multifunctional operon. pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in the tyrS-pdxY intercistronic region.

We obtained a series of Escherichia coli strains in which gapA, gapB, or both had been deleted. Delta gapA strains do not revert on glucose, while delta gapB strains grow on glycerol or glucose. We showed that gapB-encoded protein is expressed but at a very low level. Together, these results confirm the essential role for gapA in glycolysis and show that gapB is dispensable for both glycolysis and the pyridoxal biosynthesis pathway.