Control of vitamin B 6 biosynthesis in Escherichia coli

Pyridoxineless mutants of Escherichia coli B which specifically require pyridoxal or pyridoxamine for growth can be divided into classes according to their growth responses in enriched media. Members of the slowest growing class synthesize vitamin B(6) at the fastest rates when starved for pyridoxal in glycerol minimal medium. After 80 min of synthesis at 4 x 10(-10) moles of vitamin B(6) per mg of cells per hr, the rate increases four- to fivefold and continues at the new rate for several hours. The shift to the new rate is prevented by chloramphenicol, thus suggesting that a derepression mechanism exists to control vitamin B(6) synthesis in addition to the previously discovered feedback control.     

Further Characterization of Glycolaldehyde Dehydrogenase Isozymes from Escherichia coli B Involvement in Vitamin B6 Biosynthesis

Kinetic study of glycolaldehyde dehydrogenase was performed with isozymes of Escherichia coli B. The reaction equilibrium of isozyme A was estimated to lie in glycolate formation, while those of isozymes B and C were in glycolaldehyde formation. Isozyme A was released from cells with osmotic shock, while the others were not. Isozymes B and C were found in cytoplasmic fraction. Some reversal mutants derived from WG3 strain (one of vitamin B₆ auxotrophs) acquired ability to produce isozyme C. Based on these results, the non-involvement of isozyme A in vitamin B₆ biosynthesis was discussed.     

The isolation and characterization of three types of vitamin B6 auxotrophs of Escherichia coli K12.

Approximately 500 vitamin B6 auxotrophs were isolated from 18 independent cultures of Escherichia coli strain CR63. None grew in minimal medium supplemented with 2'-hydroxypyridoxine. Eighteen auxotrophs which had arisen independently were further characterized. All of them were defective in vitamin B6 synthesis rather than in an aminotransferase involved in vitamin B6 utilization. Two different phenotypes were recognized: 'Oxidase' mutants which grew only when supplied with pyridoxal or pyridoxal 5'-phosphate and 'Pre Pn' mutants which would also grow with pyridoxine or pyridoxine phosphate. "Oxidase' mutants were confined to a single linkage group, but data from interrupted mating experiments established that 'Pre Pn' mutants fall into two linkage groups which are possibly identical to pdxA and pdxB. All mutations in the in the pdxA region were allelic rather than located in two closely linked genes.     

Transport of vitamin B12 in tonB mutants of Escherichia coli.

It is known that the tonB mutation in Escherichia coli is responsible for a defect in the transport of iron chelates. These are transported by systems that involve outer membrane components. We found that tonB mutants were also deficient in the secondary, energy-dependent phase of vitamin B12 transport, although the mutants have normal levels of B12 receptors on their cell surface. In addition, tonB mutants derived from vitamin B12 auxotrophs required elevated levels of B12 for normal growth. Maltose uptake, mediated by another transport system involving an outer membrane component, was unaffected by the tonB mutation.     

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.     

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.     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.     

Biosynthesis of Vitamin B6 by a Yeast Mutant

The gradient-plate technique was employed to isolate mutants of Saccharomyces marxianus (NRRL-Y-1550) which, when grown in a synthetic culture medium, excreted about 2 mug/ml of vitamin B(6) as ascertained by microbiological assay. The major component that possessed vitamin B(6) activity was isolated by ion-exchange column chromatography and identified as pyridoxol by ultraviolet and fluorescence spectroscopy, as well as by paper chromatography and various chemical tests. Pyridoxal was also identified as one of the excreted compounds. Two other compounds that possessed vitamin B(6) activity were excreted in smaller quantities in the growth medium and have not yet been identified; they are not phosphates of vitamin B(6). The amount of vitamin B(6) excreted was not increased when the mutant was grown in the presence of various oxidation products of this vitamin. The methods and results reported here may be helpful in future studies on the biosynthesis of vitamin B(6).     

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.     

Clinical implications of the correlation between coenzyme Q10 and vitamin B6 status.

The endogenous biosynthesis of the quinone nucleus of coenzyme Q10 (CoQ10) from tyrosine is dependent on adequate vitamin B6 nutriture. Lowered blood and tissue levels of CoQ10 have been observed in a number of clinical conditions. Many of these clinical conditions are most prevalent among the elderly. Kalen et al. have shown that blood levels of CoQ10 decline with age. Similarly, Kant et al. have shown that indicators of vitamin B6 status also decline with age. Blood samples were collected from 29 patients who were not currently being supplemented with either CoQ10 or vitamin B6. Mean CoQ10 concentrations was 1.1 +/- 0.3 micrograms/ml of blood. Mean specific activities of EGOT was 0.30 +/- 0.13 mumol pyruvate/hr/10(8) erythrocytes and the mean percent saturation of EGOT with PLP was 78.2 +/- 13.9%. Means for all parameters were within normal ranges. Strong positive correlation was found between CoQ10 and the specific activity of EGOT (r = 0.5787, p < 0.001) and between CoQ10 and the percent saturation of EGOT with PLP (r = 0.4174, p < 0.024). Studies are currently in progress to determine the effect of supplementation with vitamin B6 of blood CoQ10 levels. It appears prudent to recommend that patients receiving supplemental CoQ10 be concurrently supplemented with vitamin B6 to provide for better endogenous synthesis of CoQ10 along with the exogenous CoQ10.     

Vitamin B 6 biosynthesis in Bacillus subtilis]

1) A vitamin-B6-producing mutant, BA 1, was selected by treatment of Bacillus subtilis with N-methyl-N'-nitro-N-nitrosoguanidine. Using gradient plates supplemented with the vitamin B6 antagonist isonicotinohydrazide, three mutants of BA 1 were isolated, which excrete 2-5 mg of vitamin B6/l of growth medium. 2) Mutation of the three vitamin-B6-producing strains BA 1, BA 11 and L 71 led to the isolation of 49 vitamin-B6 deficient mutants. All mutants are able to grow with pyridoxine, pyridoxal, pyridoxamine, and even with 4'-deoxypyridoxine. Glycolaldehyde or nicotinic acid do not support growth of the mutants. Some of these vitamin-B6-deficient mutants can also grow in the absence of vitamin B6, providing isoleucine is present. Others show a growth stimulation, when isoleucine is added to a medium containing a vitamin B6 compound. Isoleucine can be replaced by 3-methyl-2-oxovalerate. Cross-feeding experiments indicated a division of the mutants into two groups. Using chromatographic methods, substances which support growth of the mutants were purified, but have not yet been identified. Following the addition of 4'-deoxypyridoxine, 4'-deoxypyridoxine 5'-phosphate was isolated from the growth medium of a vitamin B6-deficient mutant. 3) Threonine dehydratase, transaminase B and transaminase C from wild-type Bacillus subtilis were compared with the enzymes from vitamin-B6-producing strains and with the enzymes from vitamin-B6-deficient mutants. Both the vitamin-B6-producing and the vitamin B6-deficient mutants show higher specific activities than wild type. In the mutant strains no multivalent repression of the threonine dehydratase and transminase B by isoleucine, leucine and valine could be demonstrated. Leucine dehydrogenase, the first enzyme of the isoleucine catabolic pathway, is constitutively produced in the vitamin-B6-producing and in the vitamin-B6-deficient mutants. In the vitamin-B6-deficient mutants, there is a correlation between growth yield in the presence of isoleucine and the specific activity of leucine dehydrogenase. In the crude extract of Bacillus subtilis no pyridoxamine-phosphate oxidase activity could be demonstrated, whereas pyridoxal kinase was readily detectable.     

An Escherichia coli K-12 tktA tktB mutant deficient in transketolase activity requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth.

We show that a tktA tktB double mutant, which is devoid of the two known transketolase isoenzymes of Escherichia coli K-12, requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth. This pyridoxine requirement can also be satisfied by 4-hydroxy-L-threonine or glycolaldehyde. These results provide direct evidence that D-erythrose-4-phosphate is a precursor of the pyridine ring of pyridoxine. In addition, they show that the two major E. coli transketolase isoenzymes are not required for the biosynthesis of D-1-deoxyxylulose, which is thought to be another precursor of pyridoxine.     

Alterations of KCl- and ATP-induced increase in [Ca2+]i in cardiomyocytes from vitamin B6 deficient rats

Although vitamin B6 deficiency is related to coronary heart disease, no information regarding changes in myocardium due to vitamin B6 deficiency is available in the literature. In view of the critical role played by Ca2+ in cellular function, we investigated alterations in [Ca2+]i induced by KCI or ATP in vitamin B6 deficient and age-matched control rats. [Ca2+]i was measured in isolated cardiomyocytes by using the Fura-2 fluorescence technique. The KC1-induced increase in [Ca2+]i was augmented in vitamin B6 deficient cardiomyocytes, whereas the ATP-induced increase in [Ca2+]i was attenuated. The specific ATP binding to sarcolemma from hearts of vitamin B6 deficient rats was decreased. A single injection of vitamin B6 (10 mg/kg) to vitamin B6 deficient animals completely reversed the KC1- or ATP-induced changes in [Ca2+]i in cardiomyocytes as well as ATP binding with sarcolemma. These results regarding altered regulation of [Ca2+]i in cardiomyocytes and sarcolemmal ATP receptors indicate myocardial abnormalities due to vitamin B6 deficiency.     

Glycolaldehyde is a precursor of pyridoxal phosphate in Escherichia coli B

Carbon-labeled glycolaldehyde prepared from [(14)C]serine was used to supply the nutritional requirement of a pyridoxineless auxotroph of Escherichia coli. Pyridoxal phosphate isolated from bacteria so grown was found to have incorporated the radioactive glycolaldehyde with little dilution. The radioactivity which was unincorporated into pyridoxal phosphate was recovered almost entirely in the culture fluid. The results establish for the first time that glycolaldehyde is indeed a natural precursor of pyridoxal phosphate or it is readily converted to such a precursor.     

Photo-induced processes in vitamin B6 compounds

In this work, we applied multi-wavelength stopped-flow spectroscopy (MSFS) to study the chemical equilibria between tautomeric or hydrated forms of various vitamin B6 compounds and the Schiff base formed by epsilon-aminocaproic acid (= 6-aminohexanoic acid) with pyridoxal 5'-phosphate at 25 degrees and variable pH. Since some of these compounds are photosensitive, we analyzed the possible occurrence of any secondary photo-induced processes under the conditions of irradiation in the MSFS equipment (continuous irradiation with light from a 75-W Xe lamp spanning the wavelength range of 200-700 nm). To determine the tautomeric composition of these compounds, the electronic absorption spectra were analyzed by means of log-normal curves. Continuous irradiation of pyridoxamine and pyridoxal 5'-phosphate over the wavelength range of 200-700 nm displaces the chemical equilibrium between the tautomeric or hydrated forms of these compounds. However, the Schiff base of epsilon-aminocaproic acid with pyridoxal 5'-phosphate is insensitive to the radiation used. The photo-induced processes detected in pyridoxamine and pyridoxal 5'-phosphate should be taken into account in examining vitamers by MSFS. In fact, these additional processes should be considered in studying the mechanism of action of vitamin B6-dependent enzymes by the MSFS technique, whenever some free vitamer may be present in solution.     

Effects of vitamin B6 supplementation in rats during lactation on vitamin B6 concentration and transaminase activities in the offspring

The aim of the present investigation was to study the effect of a varying maternal vitamin B₆ supplementation during lactation period on vitamin B₆ levels in blood, liver and total body, and on the activity of two transaminase enzymes in the offspring. Therefore, eighty female Sprague‐Dawley rats were fed a semi‐synthetic diet (0.2 mg vitamin B₆ per kg) which was supplemented during gravidity with 5 mg vitamin B₆ per kg diet. During the following lactation period the rats were assigned to one of 10 vitamin B₆ treatment groups (supplementation of 0, 3, 6, 9, 12, 15, 18, 36, 360, 3600 mg vitamin B₆ per kg diet). At day 14 of lactation the pubs of all dams were decapitated and blood, liver, and carcass were used for analysis of vitamin B₆ concentration, activities of two transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma, erythrocytes, and liver, and of haematological parameters.

While the liver and total body wet weights as well as the haematological parameters (red blood cells, haemoglobin concentration, hematocrit, middle corpuscular cell volume, middle corpuscular haemoglobin, middle corpuscular haemoglobin concentration) did not differ within the experimental groups, the present data clearly show that in blood, liver and total body of the offspring exists a slight dose‐response relationship between the maternal dietary vitamin B₆ supplementation and the vitamin B₆ concentration. Concerning the activities of the transaminases a dietary supplementation above 3mg vitamin B₆ per kg diet had no influence on the AST and ALT activities in offspring plasma. In the erythrocytes no statistical significant influence of the vitamin B₆ supplementation during lactation on the activities of AST and ALT was found. The activities of ALT and AST in liver were not consistently altered by the vitamin B₆ supplementation of the dams during lactation. In conclusion these results indicate that a minimal maternal dietary vitamin B₆ supply of 3.1 mg per kg diet is necessary with regard to health and development of their offspring. But not all of the analysed parameters as the liver and total body weights, the activities of AST and ALT in the erythrocytes, and the haematological parameters were influenced by a deficient maternal dietary vitamin B₆ supply.     

Flavin adenine dinucleotide-dependent 4-phospho-D-erythronate dehydrogenase is responsible for the 4-phosphohydroxy-L-threonine pathway in vitamin B6 biosynthesis in Sinorhizobium meliloti

The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-D-erythronate (4PE) dehydrogenase. This enzyme catalyzes the oxidation of 4PE to 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate and is clearly different in terms of cofactor requirements from the pdxB gene product of E. coli, which is known to be an NAD-dependent enzyme. Previously, we revealed that in S. meliloti IFO 14782, 4PHT is synthesized from 4-hydroxy-l-threonine and that this synthesis starts with glycolaldehyde and glycine. However, in this study, we identified a second 4PHT pathway in S. meliloti that originates exclusively from glycolaldehyde (the major pathway). Based on the involvement of 4PE in the 4PHT pathway, the incorporation of different samples of 13C-labeled glycolaldehyde into pyridoxine molecules was examined using 13C nuclear magnetic resonance spectroscopy. On the basis of the spectral analyses, the synthesis of 4PHT from glycolaldehyde was hypothesized to involve the following steps: glycolaldehyde is sequentially metabolized to D-erythrulose, D-erythrulose 4-phosphate, and D-erythrose 4-phosphate by transketolase, kinase, and isomerase, respectively; and D-erythrose 4-phosphate is then converted to 4PHT by the conventional three-step pathway elucidated in E. coli, although the mechanism of action of the enzymes catalyzing the first two steps is different.