Vitamin B(6) salvage enzymes: mechanism, structure and regulation

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats.

The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

Effect of tryptophan metabolites on the activities of rat liver pyridoxal kinase and pyridoxamine 5-phosphate oxidase in vitro.

Pyridoxal kinase was purified 4760-fold from rat liver. The Km values for pyridoxine and pyridoxal were 120 and 190 microM respectively, and pyridoxine showed substrate inhibition at above 200 microM. Pyridoxamine 5-phosphate oxidase was also purified 2030-fold from rat liver, and its Km values for pyridoxine 5-phosphate and pyridoxamine 5-phosphate were 0.92 and 1.0 microM respectively. Pyridoxine 5-phosphate gave a maximum velocity that was 5.6-fold greater than with pyridoxamine 5-phosphate and showed strong substrate inhibition at above 6 microM. Among the tryptophan metabolites, picolinate, xanthurenate, quinolinate, tryptamine and 5-hydroxytryptamine inhibited pyridoxal kinase. However, pyridoxamine 5-phosphate oxidase could not be inhibited by tryptophan metabolites, and on the contrary it was activated by 3-hydroxykynurenine and 3-hydroxyanthranilate. Regarding the metabolism of vitamin B-6 in the liver, the effects of tryptophan metabolites that were accumulated in vitamin B-6-deficient rats after tryptophan injection were discussed.     

On the inhibitory activity of 4-vinyl analogues of pyridoxal: enzyme and cell culture studies.

Analogues of pyridoxal and of pyridoxal phosphate in which the 4-CHO group is replaced with CH = CH2 were synthesized and were found to be potent inhibitors of pyridoxal kinase and pyridoxine phosphate oxidase of rat liver. They also inhibited the growth of mouse Sarcoma 180 and mammary adenocarcinoma TA3 in cell culture. Saturation of the vinyl double bond, replacement of the 5-CH2OH with methyl, methylation of the phenolic hydroxyl, or conversion to the N-oxide resulted in diminution or loss of all these activities. Similarly, the introduction of a beta-methyl group into the vinyl analogues of pyridoxal reduced all these inhibitory activities. The 4-vinyl anatogue of pyridoxal was shown to be a substrate of pyridoxal kinase and the product a potent inhibitor of pyridoxine oxidase, competing with pyridoxal phosphate. The affinity of this phosphorylated pyridoxal analogue to some apoenzymes varied greatly, indicating striking differences among the cofactor binding sites of these enzymes. The growth inhibitory effects of these analogues on cells in culture correlated well with their effects on pyridoxal kinase and pyridoxine phosphate oxidase in cell-free systems.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats

The metabolism of [6-³H]pyridoxine · HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding adlititum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 μCi of [6-³H]pyridoxine · HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H⁺) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days.In vitamin B-6-deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5′-P and pyridoxal 5′-P reached 36 and 44% levels, respectively. Pyridoxine 5′-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5′-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5′-P was falling, pyridoxamine 5′-P was linearly synthesized reaching a plateau level (62%) in 5 days which also remained unchaged through 7 days. Some pyridoxal was also formed (7% at 1 h) which by 12 h had dropped to a plateau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5′-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [³H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5′-P synthesis, and (c) a continuouus synthesis (and accumulation) of pyridoxamine 5′-P which is not utilized or further metabolized.     

Possible influence of gonadotrophins on formation in vivo of pyridoxal phosphate

FSH administered to normal rats increased the activity of pyridoxine phosphate oxidase of both liver and kidney and, consequently, pyridoxal phosphate levels in these tissues were elevated. LH administration, on the other hand, decreased the activity of pyridoxine phosphate oxidase, resulting in diminished pyridoxal phosphate level in the tissues. The stimulatory effect of FSH on the activity of liver and kidney pyridoxine phosphate oxidase was not observed in castrated-adrenalectomised rats unless supplemented with cortisone and testosterone, respectively. Puromycin treatment prevented the FSH-induced rise in the activity of liver and kidney pyridoxine phosphate oxidases. It is suggested that FSH stimulates the activity of liver and kidney pyridoxine phosphate oxidase by increasing the synthesis of apoproteins of the enzyme, and the effect of FSH on liver is dependent on the presence of adrenal corticoids while the presence of testosterone is a prerequisite for the FSH to have its effect on kidney pyridoxine phosphate oxidase.     

Vitamin B6 deficiency decreases the glucose utilization in cognitive brain structures of rats

The effects of vitamin B(6) deficiency on metabolic activities of brain structures were studied. Male Sprague-Dawley weanling rats received one of the following diets: (1) 7 mg pyridoxine HCl/kg (control group); (2) 0 mg pyridoxine HCl/kg (vitamin B(6)-deficient group); or (3) 7 mg pyridoxine HCl/kg with food intake restricted in quantity to that consumed by the deficient group (pair-fed control group). After 8 weeks of dietary treatment, rats in all three groups received an intravenous injection of 2-deoxy-[(14)C] glucose (100 microCi/kg). Vitamin B(6) status was evaluated by plasma pyridoxal 5'-phosphate concentrations. The vitamin B(6)-deficient group had significantly lower levels of plasma pyridoxal 5'-phosphate than did the control and pair-fed groups. The local cerebral glucose utilization rates in structures of the limbic system, basal ganglia, sensory motor system, and hypothalamic system were determined. The local cerebral glucose utilization rates in each of the four brain regions in the deficient animals were approximately 50% lower (P < 0.05) than in the control group. Results of the present study suggest that serious cognitive deficit may occur in vitamin B(6)-deficient animals.     

Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity.

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1'-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.     

The conversion of pyridoxine phosphate into pyridoxal phosphate in Escherichia coli

1. Evidence is presented for the presence of pyridoxine phosphate oxidase in aqueous extracts of Escherichia coli. Some comparison is made with pyridoxamine phosphate oxidase. 2. Isoniazid and iproniazid were found to combine with pyridoxal phosphate, but isoniazid did not combine with either pyridoxamine phosphate or pyridoxine phosphate. Both oxidase activities were somewhat inhibited by benzylamine and putrescine, but not by phenethylamine or cadaverine. 3. The significance of pyridoxine phosphate oxidase in cell metabolism is discussed.     

Glutathione reductase activity and pyridoxine (pyridoxamine) phosphate oxidase activity in the red cell

The red-cell enzymes, glutathione reductase (FAD-dependent) and pyridoxine (pyridoxamine) phosphate oxidase (FMN-dependent), were studied in control subjects. The wide range the glutathione reductase activity correlated inversely with the percentage stimulation by FAD added in vitro, and with pyridoxine (pyridoxamine) phosphate oxidase activity. Both enzymes were stimulated after ingestion of riboflavin. The results support the suggestion that the rate of metabolism of riboflavin in the red cell controls the activity of both enzymes, and the rate of red-cell metabolism of vitamin B-6.     

Pyridoxine kinase in Plasmodium lophurae and duckling erythrocytes.

Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent Km for pyridoxine of the parasite enzyme was 6.6 times 10(-5) M whereas the host red cell enzyme Km was 1.9 times 10(-6) M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent Ki of 1.5 times 10(-6) and 8.6 times 10(-6) M, respectively. These results suggest that the vitamin B6 metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.     

Identification of pyridoxine 3-sulfate, pyridoxal 3-sulfate, and N-methylpyridoxine as major urinary metabolites of vitamin B6 in domestic cats

Preliminary studies of vitamin B6 metabolism in three adult domestic cats detected very little pyridoxic acid in the urine. At oral doses of 49 to 490 mumol of [14C]pyridoxine hydrochloride, 50% of the excreted dose occurred as pyridoxine 3-sulfate and 25% as N-methylpyridoxine. The identity of these two metabolites was confirmed by isolation from urine and comparison with known compounds. A third compound was identified as pyridoxal 3-sulfate on the basis of chromatographic behavior and fluorescent properties before and after hydrolysis. At pyridoxine intakes of 0.97 mumol/day, the concentration of pyridoxal 3-sulfate in the urine sometimes exceeded the concentration of pyridoxine 3-sulfate. Pyridoxic acid remained a minor urinary metabolite at pyridoxine intakes ranging from 0.97 to 490 mumol/day. Although sulfation of phenol groups and methylation of the ring nitrogen are well-known detoxication reactions, this appears to be the first time such reactions have been observed in normal metabolism of vitamin B6. These observations provide further evidence of the diversity of vitamin B6 metabolism between species. While such diversity complicates the extrapolation of data from animal studies to humans, it does provide a variety of models for examining the influences of various factors on vitamin B6 metabolism.     

Interrelationships between pyridoxal phosphate and pyridoxal kinase in rabbit brain

—An inverse relationship was demonstrable between the concentration of pyridoxal phosphate and the activity of pyridoxal kinase in rabbit brain. The administration of pyridoxine elevated the concentration of pyridoxal phosphate and decreased the activity of pyridoxal kinase. Conversely, the administration of deoxypyridoxine decreased the concentration of pyridoxal phosphate and increased the activity of pyridoxal kinase. The increase in the activity of pyridoxal kinase by deoxypyridoxine was blocked by actinomycin D or puromycin. These results were interpreted to indicate that the tissue availability of pyridoxal phosphate regulated the activity of pyridoxal kinase.     

Vitamin B6 influences glucocorticoid receptor-dependent gene expression

We have examined the influence of intracellular vitamin B6 concentration on glucocorticoid receptor function in HeLa S3 cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. CAT activity is induced from this plasmid specifically by glucocorticoid hormones in a glucocorticoid receptor-dependent manner. The intracellular concentration of pyridoxal phosphate, the physiologically active form of the vitamin, was elevated by supplementation of the culture medium with the synthesis precursor pyridoxine and lowered by exposure to the pyridoxal phosphate synthesis inhibitor 4-deoxypyridoxine. Analysis of glucocorticoid responsiveness revealed that elevated concentrations of intracellular pyridoxal phosphate suppressed the amount of glucocorticoid-induced CAT activity whereas moderate deficiency enhanced the level of glucocorticoid receptor-mediated gene expression. In contrast, modulation of the intracellular pyridoxal phosphate concentration had no effect on either basal CAT activity derived from cells not stimulated with dexamethasone or on CAT activity derived from two glucocorticoid-insensitive reporter plasmids. The modulatory effects of pyridoxal phosphate concentration occur without changes in glucocorticoid receptor mRNA levels, glucocorticoid receptor protein concentration, or the steroid binding capacity of the receptor. These observations demonstrate that vitamin B6 selectively influences glucocorticoid receptor-dependent gene expression through a novel mechanism that does not involve alterations in glucocorticoid receptor concentration or ligand binding capacity.     

The effect of vitamin B6 deficiency on cytotoxic immune responses of T cells, antibodies, and natural killer cells, and phagocytosis by macrophages

The effect of vitamin B6 on cytotoxic immune responses of T cells, natural killer (NK) cells, cytotoxic antibody production, and macrophage phagocytosis was assessed in 5-week-old female C57B1/6 mice. Mice were fed 20% casein diets with pyridoxine (PN) added at 7, 1, 0.1, or 0 mg/kg diet, which represents 700, 100, 10, and 0% of requirement, respectively. Compared to mice fed 7 or 1 mg PN diet, animals fed 0 or 0.1 mg PN diet showed significantly reduced primary splenic and peritoneal T-cell-mediated cytotoxicity (CMC). Animals fed 0 mg PN diet also showed significantly depressed secondary T CMC of splenic and peritoneal lymphocytes against P815 tumor cells. Complement-dependent antibody-mediated cytotoxicity against P815 cells, phagocytosis of SRBC by macrophages, and native and interferon-induced NK cell activities against YAC cells were not affected by the level of vitamin B6 intake. The percentage of macrophages present in the peritoneal exudate cells was increased in animals fed the 0 mg PN diet. The immune responses were not enhanced or altered by the excess intake of vitamin B6 (7 mg PN). It appears that vitamin B6 is an essential nutrient for maintenance of normal T-cell function in vivo.     

Antitumor effect of vitamin B6 and its mechanisms

Epidemiological studies have reported an inverse association between vitamin B(6) intake and colon cancer risk. Our recent study has been conducted to examine the effect of dietary vitamin B(6) on colon tumorigenesis in mice. Mice were fed diets containing 1, 7, 14 or 36 mg/kg pyridoxine for 22 weeks, and given a weekly injection of azoxymethane (AOM) for the initial 10 weeks. Compared with the 1 mg/kg pyridoxine diet, 7, 14 and 35 mg/kg pyridoxine diets significantly suppressed the incidence and number of colon tumors, colon cell proliferation and expressions of c-myc and c-fos proteins. Supplemental vitamin B(6) lowered the levels of colonic 8-hydroxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE, oxidative stress markers) and inducible nitric oxide (NO) synthase protein. In an ex vivo serum-free matrix culture model using rat aortic ring, supplemental pyridoxine and pyridoxal 5'-phosphate (PLP) had antiangiogenic effect. The results suggest that dietary vitamin B(6) suppresses colon tumorigenesis by reducing cell proliferation, oxidative stress, NO production and angiogenesis.     

Vitamin B6 metabolism in microbes and approaches for fermentative production

Vitamin B6 is a designation for the six vitamers pyridoxal, pyridoxine, pyridoxamine, pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate, and pyridoxamine. PLP, being the most important B6 vitamer, serves as a cofactor for many proteins and enzymes. In contrast to other organisms, animals and humans have to ingest vitamin B6 with their food. Several disorders are associated with vitamin B6 deficiency. Moreover, pharmaceuticals interfere with metabolism of the cofactor, which also results in vitamin B6 deficiency. Therefore, vitamin B6 is a valuable compound for the pharmaceutical and the food industry. Although vitamin B6 is currently chemically synthesized, there is considerable interest on the industrial side to shift from chemical processes to sustainable fermentation technologies. Here, we review recent findings regarding biosynthesis and homeostasis of vitamin B6 and describe the approaches that have been made in the past to develop microbial production processes. Moreover, we will describe novel routes for vitamin B6 biosynthesis and discuss their potential for engineering bacteria that overproduce the commercially valuable substance. We also highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies to circumvent these limitations.     

Effects of pyridoxine supplementation on blood glucocorticoids level, aspartate aminotransferase activity and glucose tolerance pattern under acute hypoxic stress

Studies were conducted on male adult rabbits to find out the changes in blood glucocorticoid levels along with the changes in aspartate aminotransferase activity in blood and the role of pyridoxine on the glucose tolerance pattern under hypoxic stress. Hypoxic stress was produced by exposing the animals to a simulated altitude of 7,000 m for 6 h. In the first set of experiments 10 rabbits were used. Blood haemoglobin level, plasma and erythrocyte glucocorticoid levels and erythrocyte GOT activity were measured just before and after the exposure to hypoxia. Erythrocyte GOT activity was measured both without and with 50 mg of pyridoxal phosphate addition to the incubation mixture. Glucocorticoid levels in plasma increased by 11% whereas in erythrocytes the increase was 55% after hypoxia. Percent stimulation of erythrocyte GOT activity with pyridoxal phosphate before exposure to hypoxia was 180% but increased to 321% after exposure. In the second set of experiments another 10 rabbits were used. First they were exposed to hypoxia without pyridoxine hydrochloride feeding and then after 7 days with 3 mg of pyridoxine hydrochloride feeding. For glucose tolerance tests the animals were fed with 1 g of glucose immediately after the hypoxic exposures. Plasma reduced glutathione (GSH), LDH and ICDH activities increased and GOT activity was depressed after hypoxic stress, but when the animals were fed pyridoxine hydrochloride prior to the exposure the enzyme activities remained unaltered after hypoxic stress. Pyridoxine hydrochloride did not alter the pattern of glucose tolerance after hypoxic stress.