Biosynthesis of Vitamin B6 in Rhizobium: In Vitro Synthesis of Pyridoxine from 1-Deoxy-D-xylulose and 4-Hydroxy-L-threonine

Pyridoxine (vitamin B₆) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD⁺, NADP⁺, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD⁺ for formation of pyridoxine 5′-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.     

Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis

Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.     

Biosynthesis of vitamin B(6) in rhizobium

The biosynthetic pathway of pyridoxol (vitamin B(6)) in Rhizobium was clarified by studies on the incorporation of (13)C- or (15)N-labeled precursors into pyridoxol or its biosynthetic intermediates. Pyridoxol was formed by ring closure of two compounds, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. The former was formed from D-glyceraldehyde and pyruvate through decarboxylation of pyruvate, and the latter from glycine and glycolaldehyde.     

Hypertension,calcium channel and pyridoxine (vitamin B6)

The moderately pyridoxine (vitamin B₆)-deficient male rat was introduced by us as an animal model (B6DHT) for the study of hypertension. Hypertension in this rat is associated with increased sympathetic stimulation. Arterial segments from B6DHT rats maintained a higher resting tone. The influx of ⁴⁵calcium into intracellular compartment of the vascular smooth muscle of the caudate artery of B6DHT rats was also enhanced. Administration of pyridoxine attenuated the hypertension in B6DHT rats as well as in genetic or dietary-induced moderately hypertensive conditions such as in the Zucker obese rat and sucrose or low calcium-fed rats. However, pyridoxine did not have any effect or the spontaneously hypertensive rat. All classes of calcium channel blockers were effective in lowering the systolic blood pressure of B6DHT rats. The increased in vitro influx of⁴⁵ calcium into intracellular compartment of artery segments of B6DHT rats as well as the BAY K 8644-induced influx of⁴⁵ calcium into artery segments from normal rats were blocked by pyridoxal phosphate as well as by dihydropyridine-sensitive calcium channel blockers (DHP). Pyridoxal phosphate (PLP) in vitro enhances the binding of calcium channel antagonists to membrane preparations from vascular tissue. PLP corrects the membrane abnormality in responsive hypertensive conditions and thus, could be an endogenous modulator of DHP - sensitive calcium channels.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

Role of vitamin B 6 biosynthetic rate in the study of vitamin B 6 synthesis in Escherichia coli

Nutritional auxotrophs of Escherichia coli synthesize vitamin B(6) compounds at a rate of 1 x 10(-10) to 2 x 10(-10) moles per hr per mg (dry weight) of cells when they are suspended in minimal medium lacking their required nutrients. A few auxotrophs have been found to stop or reduce vitamin B(6) synthesis during such an experiment. These include thiamineless, citrate synthaseless, and pyridoxineless mutants as well as mutants which require four carbon compounds for growth. Glycolaldehyde was found to restore vitamin B(6) synthesis in the last named of these mutants without restoring normal growth. A class of pyridoxineless mutants which responded with normal growth to 0.4 mm glycolaldehyde or 0.15 x 10(-3) mm pyridoxol was also found. The results suggest that a thiamine pyrophosphate-requiring step as well as glycolaldehyde may be involved in pyridoxal phosphate biosynthesis.     

Evolution of vitamin B6 (pyridoxine) metabolism by gain and loss of genes

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.     

Multistate binding in pyridoxine 5'-phosphate synthase: 1.96 A crystal structure in complex with 1-deoxy-D-xylulose phosphate

We report the 1.96 A crystal structure of pyridoxine 5'-phosphate synthase (PdxJ) in complex with 1-deoxy-D-xylulose phosphate (dXP). The octameric enzyme possesses eight distinct binding sites, and three different binding states are observed. The observation of these three states supports a mechanism in which precise conformational changes of a peptide loop and groups of active site residues modulate binding and specificity. The differences in protein conformation when one or two substrates are bound can be correlated with a condensation mechanism that leads productively to the formation of pyridoxine 5'-phosphate (PNP). "Snapshots" of the progression from the apo form to a singly occupied "transitional binding" state and, subsequently, to a fully occupied, reactive state are revealed and indicate how the enzyme structure can be related to a plausible catalytic mechanism and, moreover, to favorable energetics of reaction.     

Biosynthesis of vitamin B2.

GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, the first committed intermediate in the biosynthesis of riboflavin. The enzyme was shown to contain one zinc ion per subunit. Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analog, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate. However, the mutant proteins retained the capacity to release pyrophosphate from GTP and from the formamide-type intermediate analog. The data suggest that the enzyme catalyzes an ordered reaction in which the hydrolytic release of pyrophosphate precedes the hydrolytic attack of the imidazole ring. Ring opening and formate release are both dependent on a zinc ion acting as a Lewis acid, which activates the two water molecules involved in the sequential hydrolysis of two carbon-nitrogen bonds.     

Biosynthesis of homocysteine sulfinic acid in the vitamin B6-deficient rat.

The formation of L-homocysteine sulfinic acid in the rat was examined in vivo and in vitro. This compound was rarely found, in trace amounts, in the urine and could not be detected in the tissues of various animal species studied. When L-homocysteine or methionine was given orally or intraperitoneally to rats deficient in vitamine B6, the level of homocysteine sulfinic acid in the urine increased and its synthesis could be detected in the liver.