PYRIDOX(AM)INE 5'-PHOSPHATE OXIDASE3 of Arabidopsis thaliana maintains carbon/nitrogen balance in distinct environmental conditions.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization.

BACKGROUND: Microbial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory. RESULTS: In this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation. CONCLUSION: Our results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.

4'-Deoxypyridoxine disrupts vitamin B6 homeostasis in Escherichia coli K12 through combined inhibition of cumulative B6 uptake and PLP-dependent enzyme activity.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B6 as previously claimed and that it is toxic in several conditions where vitamin B6 homeostasis is affected, such as in a B6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4'-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

Mechanism of Pyridoxine 5'-Phosphate Accumulation in Pyridoxal 5'-Phosphate-Binding Protein Deficiency.

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. This accumulated PNP could affect diverse metabolic systems through the inhibition of some PLP-dependent enzymes. In this study, we investigated the as-yet-unclear mechanism of intracellular accumulation of PNP due to the loss of PLPBP protein encoded by yggS in E. coli. Genetic studies using several PLPBP-deficient strains of E. coli lacking a known enzyme(s) in the de novo or salvage pathways of vitamin B6, including pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies of the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa. Here, we show that disruption of PLPBP perturbs PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. IMPORTANCE A PLP-binding protein (PLPBP) from the conserved COG0325 family has recently been recognized as a key player in vitamin B6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of this accumulation has been poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and that its disruption may lead to the accumulation of PNP in PLPBP deficiency.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Preventing Vitamin B6-Related Neurotoxicity.

BACKGROUND: Vitamin B6 is essential for life and plays a critical role in many biochemical and physiological processes in the human body. The term B6 collectively refers to 6 water-soluble vitamers, and only the pyridoxal 5'-phosphate (PLP) serves as the biologically active form. A plasma PLP concentration above 30 nmol/L (7.4 µg/L) is indicative of an adequate vitamin B6 status for all age and sex groups. The currently recommended daily allowance of B6 (1.5-2 mg/d) from dietary sources frequently results in inadequate B6 status (<20 nmol/L or 5 µg/L) in many elderly patients and patients with comorbid conditions. PLP-based supplements are preferred and should be administered weekly in low doses (50-100 mg) to maintain a stable serum PLP level between 30 and 60 nmol/L or 7.4 and 15 µg/L. AREAS OF UNCERTAINTY: It is challenging for physicians to prescribe a safe dose of B6 supplements because of the narrow therapeutic index. The association between elevated levels of pyridoxine and neuropathy is not well established. PLP-based supplements are shown to be least neurotoxic, but further clinical trials are needed to establish the long-term safety in high doses. DATA SOURCES: PubMed search of randomized control trials and meta-analyses. THERAPEUTIC OPINION: Plasma B6 levels should be ordered as a part of workup of any unexplained anemia before labeling as "anemia of chronic disease." B6 supplementation is also crucial in the management of chronic Mg deficiency resistant to therapy. When B6 is administered daily in supraphysiologic doses, there is a potential for the development of neurotoxicity (typically at levels >100 nmol/L or 25 µg/L). PLP-based supplements are preferred over pyridoxine supplements because of minimal neurotoxicity observed in neuronal cell viability tests. Since B6 metabolites have a long half-life, weekly administration is preferred over daily use to prevent toxicity.

Pyridoxine Has a Potential to Prevent the Appearance of Pigmented Spots: Effects on the Phagocytosis and Differentiation of Keratinocytes.

Pyridoxine (VB6) is a vitamin that is essential to maintain the homeostasis of the human body by contributing to various metabolic reactions. In the skin, although some studies have shown that VB6 is involved in regulating homeostasis through the attenuation of intracellular oxidative stress, there are few reports regarding the effects of VB6 on the prevention or improvement of skin aging. Thus, we conducted this study to determine the potential anti-skin pigmentation effect of VB6 focusing on the phagocytosis of melanosomes (MSs) by keratinocytes. The phagocytosis of MSs by keratinocytes is activated by oxidative stress and is an important factor of skin pigmentation and the eventual appearance of pigmented spots. First, we confirmed the antioxidant property of VB6 that enhanced the expression of several intracellular antioxidants via nuclear erythroid factor 2-related factor 2 (Nrf2). Although the incorporation of fluorescent beads (FBs), which are used as pseudo-MSs, into keratinocytes was increased under higher oxidation conditions caused by UVB and by the depletion of intracellular glutathione, treatment with VB6 suppressed the increased incorporation of FBs into those keratinocytes via Nrf2 activation. Furthermore, VB6 restored the decreased expression of differentiation marker proteins in keratinocytes caused by FB incorporation. Taken together, the results show that VB6 has the potential to prevent the appearance of pigmented spots by suppressing the activation of phagocytosis in keratinocytes caused by oxidative stress, and by restoring the differentiation of keratinocytes disrupted by FB incorporation.

Role of the conserved pyridoxal 5'-phosphate-binding protein YggS/PLPBP in vitamin B6 and amino acid homeostasis.

The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5'-phosphate (PLP)-binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6-dependent epilepsy in humans. In Escherichia coli, disruption of this protein-encoded by yggS-perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5'-phosphate (PNP) in various organisms. In the yggS-deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein.

Construction and applications of a B vitamin genetic resource for investigation of vitamin-dependent metabolism in maize.

The B vitamins provide essential co-factors for central metabolism in all organisms. In plants, B vitamins have surprising emerging roles in development, stress tolerance and pathogen resistance. Hence, there is a paramount interest in understanding the regulation of vitamin biosynthesis as well as the consequences of vitamin deficiency in crop species. To facilitate genetic analysis of B vitamin biosynthesis and functions in maize, we have mined the UniformMu transposon resource to identify insertional mutations in vitamin pathway genes. A screen of 190 insertion lines for seed and seedling phenotypes identified mutations in biotin, pyridoxine and niacin biosynthetic pathways. Importantly, isolation of independent insertion alleles enabled genetic confirmation of genotype-to-phenotype associations. Because B vitamins are essential for survival, null mutations often have embryo lethal phenotypes that prevent elucidation of subtle, but physiologically important, metabolic consequences of sub-optimal (functional) vitamin status. To circumvent this barrier, we demonstrate a strategy for refined genetic manipulation of vitamin status based on construction of heterozygotes that combine strong and hypomorphic mutant alleles. Dosage analysis of pdx2 alleles in endosperm revealed that endosperm supplies pyridoxine to the developing embryo. Similarly, a hypomorphic bio1 allele enabled analysis of transcriptome and metabolome responses to incipient biotin deficiency in seedling leaves. We show that systemic pipecolic acid accumulation is an early metabolic response to sub-optimal biotin status highlighting an intriguing connection between biotin, lysine metabolism and systemic disease resistance signaling. Seed-stocks carrying insertions for vitamin pathway genes are available for free, public distribution via the Maize Genetics Cooperation Stock Center.

Pyridoxine stimulates filaggrin production in human epidermal keratinocytes.

Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

Submerged fermentation of Ginkgo biloba seed powder using Eurotium cristatum for the development of ginkgo seeds fermented products.

BACKGROUND: Ginkgo biloba seeds are well known for the significant curative effects on relieving cough and asthma. However, the development of products from ginkgo seeds still falls behind at present, resulting in a great waste of ginkgo seeds' resource. In this work, submerged fermentation of ginkgo seed powder using Eurotium cristatum was studied to investigate its feasibility as a new processing method. RESULTS: To promote the growth of E. cristatum, the optimum fermentation medium was 80.0 g L-1 of ginkgo seed powder with addition of 5.0 g L-1 calcium chloride (CaCl2 ), 4.0 g L-1 magnesium sulfate (MgSO4 ), 1.25 g L-1 zinc sulfate (ZnSO4 ) and 0.65 g L-1 iron(II) sulfate (FeSO4 ). The optimum fermentation conditions were pH 5.8 ± 0.1, inoculum size 5.1 × 106 CFU mL-1 , liquid medium volume 100 mL in 250-mL Erlenmeyer flask and fermentation 4 days. Through fermentation, the production of lovastatin in fermentation broth could reach up to 32.97 ± 0.17 µg mL-1 and the total antioxidant capacity was improved by more than two-fold. In addition, 40.15% of the ginkgotoxin in ginkgo seed powder was degraded while the entire degradation of ginkgolic acids was obtained. Moreover, fermented ginkgo seed powder suspension presented pleasant fragrances, and the activities of amylase and protease were enhanced to 11.30 ± 0.10 U mL-1 and 23.01 ± 0.20 U mL-1 , respectively. CONCLUSIONS: Submerged fermentation using E. cristatum could significantly enhance the functional value and safety of ginkgo seed powder, and had great potential to become a novel processing method for the development of ginkgo seeds fermented products. © 2020 Society of Chemical Industry.

Inherited Disorders of Lysine Metabolism: A Review.

Lysine is an essential amino acid, and inherited diseases of its metabolism therefore represent defects of lysine catabolism. Although some of these enzyme defects are not well described yet, glutaric aciduria type I (GA1) and antiquitin (2-aminoadipic-6-semialdehyde dehydrogenase) deficiency represent the most well-characterized diseases. GA1 is an autosomal recessive disorder due to a deficiency of glutaryl-CoA dehydrogenase. Untreated patients exhibit early onset macrocephaly and may present a neurological deterioration with regression and movement disorder at the time of a presumably "benign" infection most often during the first year of life. This is associated with a characteristic neuroimaging pattern with frontotemporal atrophy and striatal injuries. Diagnosis relies on the identification of glutaric and 3-hydroxyglutaric acid in urine along with plasma glutarylcarnitine. Treatment consists of a low-lysine diet aiming at reducing the putatively neurotoxic glutaric and 3-hydroxyglutaric acids. Additional therapeutic measures include administration of l-carnitine associated with emergency measures at the time of intercurrent illnesses aiming at preventing brain injury. Early treated (ideally through newborn screening) patients exhibit a favorable long-term neurocognitive outcome, whereas late-treated or untreated patients may present severe neurocognitive irreversible disabilities. Antiquitin deficiency is the most common form of pyridoxine-dependent epilepsy. α-Aminoadipic acid semialdehyde (AASA) and Δ-1-piperideine-6-carboxylate (P6C) accumulate proximal to the enzymatic block. P6C forms a complex with pyridoxal phosphate (PLP), a key vitamer of pyridoxine, thereby reducing PLP bioavailability and subsequently causing epilepsy. Urinary AASA is a biomarker of antiquitin deficiency. Despite seizure control, only 25% of the pyridoxine-treated patients show normal neurodevelopment. Low-lysine diet and arginine supplementation are proposed in some patients with decrease of AASA, but the impact on neurodevelopment is unclear. In summary, GA1 and antiquitin deficiency are the 2 main human defects of lysine catabolism. Both include neurological impairment. Lysine dietary restriction is a key therapy for GA1, whereas its benefits in antiquitin deficiency appear less clear.

The regulatory effects of pyridoxine deficiency on the grass carp (Ctenopharyngodon idella) gill barriers immunity, apoptosis, antioxidant, and tight junction challenged with Flavobacterium columnar.

The effects of dietary pyridoxine (PN) on the gill immunity, apoptosis, antioxidant and tight junction of grass cap (Ctenopharyngodon idella) were investigated in this study. Fish were fed semi-purified diets containing graded levels of PN for 10 weeks, and then challenged with Flavobacterium columnare by bath immersion exposure for 3 days. The results indicated that compared with the optimal PN level, PN deficiency resulted in a decline in the antimicrobial compound production of gill. In addition, PN deficiency up-regulated the pro-inflammatory cytokines and down-regulated the anti-inflammatory cytokines gene expression, which might be associated with the enhanced nuclear factor κB p65 and the inhibited target of rapamycin signalling pathways, respectively, suggesting that PN deficiency could impair gill immune barrier function. Furthermore, PN deficiency (1) induced cell apoptosis, which may be partly associated with the (apoptotic protease activating factor-1, Bcl-2 associated X protein)/caspase-9 and c-Rel/tumor necrosis factor α (rather than FasL)/caspase-8 mediated apoptosis pathway. (2) Inhibited Kelch-like ECH-associating protein 1a/NF-E2-related factor 2 mRNA expression, decreased the mRNA expression and activities of antioxidant enzymes, increased the levels of reactive oxygen species, protein carbonyl and malondialdehyde. (3) Increased the mRNA expression level of myosin light chain kinase, which may be result in the down-regulation of tight junction complexes such as zonula occludens 1, occludin and claudins (expect claudin-12 and claudin-15). These results suggest that PN deficiency could impair gill physical barrier function. In summary, dietary PN deficiency could cause the impairment of gill barrier function associated with immunity, apoptosis, antioxidant and tight junction, which may result in the increased the susceptibility of fish to pathogenic bacteria. Moreover, based on the gill rot morbidity, LZ activity and MDA content, the dietary PN requirements for grass cap were estimated to be 4.85, 4.78 and 4.77 mg kg-1 diet, respectively.

Pyridoxine Deficiency Exacerbates Neuronal Damage after Ischemia by Increasing Oxidative Stress and Reduces Proliferating Cells and Neuroblasts in the Gerbil Hippocampus.

We investigated the effects of pyridoxine deficiency on ischemic neuronal death in the hippocampus of gerbil (n = 5 per group). Serum pyridoxal 5'-phosphate levels were significantly decreased in Pyridoxine-deficient diet (PDD)-fed gerbils, while homocysteine levels were significantly increased in sham- and ischemia-operated gerbils. PDD-fed gerbil showed a reduction in neuronal nuclei (NeuN)-immunoreactive neurons in the medial part of the hippocampal CA1 region three days after. Reactive astrocytosis and microgliosis were found in PDD-fed gerbils, and transient ischemia caused the aggregation of activated microglia in the stratum pyramidale three days after ischemia. Lipid peroxidation was prominently increased in the hippocampus and was significantly higher in PDD-fed gerbils than in Control diet (CD)-fed gerbils after ischemia. In contrast, pyridoxine deficiency decreased the proliferating cells and neuroblasts in the dentate gyrus in sham- and ischemia-operated gerbils. Nuclear factor erythroid-2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF) levels also significantly decreased in PDD-fed gerbils sham 24 h after ischemia. These results suggest that pyridoxine deficiency accelerates neuronal death by increasing serum homocysteine levels and lipid peroxidation, and by decreasing Nrf2 levels in the hippocampus. Additionally, it reduces the regenerated potentials in hippocampus by decreasing BDNF levels. Collectively, pyridoxine is an essential element in modulating cell death and hippocampal neurogenesis after ischemia.

Pyridoxine and pancreatic acinar cells: transport physiology and effect on gene expression profile.

Pyridoxine (vitamin B6), an essential micronutrient for normal cell physiology, plays an important role in the function of the exocrine pancreas. Pancreatic acinar cells (PACs) obtain vitamin B6 from circulation, but little is known about the mechanism involved in the uptake process; limited information also exists on the effect of pyridoxine availability on the gene expression profile in these cells. We addressed both these issues in the current investigation using mouse-derived pancreatic acinar 266-6 cells (PAC 266-6) and human primary PACs (hPACs; obtained from organ donors), together with appropriate physiological and molecular (RNA-Seq) approaches. The results showed [3H]pyridoxine uptake to be 1) pH and temperature (but not Na+) dependent, 2) saturable as a function of concentration, 3) cis-inhibited by unlabeled pyridoxine and its close structural analogs, 4) trans-stimulated by unlabeled pyridoxine, 5) regulated by an intracellular Ca2+/calmodulin-mediated pathway, 6) adaptively-regulated by extracellular substrate (pyridoxine) availability, and 7) negatively impacted by exposure to cigarette smoke extract. Vitamin B6 availability was found (by means of RNA-Seq) to significantly (FDR < 0.05) modulate the expression profile of many genes in PAC 266-6 cells (including those that are relevant to pancreatic health and development). These studies demonstrate, for the first time, the involvement of a regulatable and specific carrier-mediated mechanism for pyridoxine uptake by PACs; the results also show that pyridoxine availability exerts profound effects on the gene expression profile in mammalian PACs.

Hydrogen sulphide production by Saccharomyces cerevisiae UCD 522 in a synthetic grape juice medium deficient of thiamin (vitamin B1 ) and/or pyridoxine (vitamin B6 ).

The impacts of thiamin and pyridoxine along with YAN on alcoholic fermentation and hydrogen sulphide production by Saccharomyces cerevisiae were studied. Using a synthetic grape juice medium, three fermentation trials were conducted; (i) 2 × 3 factorial design with thiamin (0, 0·2, or 0·5 mg l-1 ) and YAN (60 or 250 mg l-1 ) as variables, (ii) 2 × 3 factorial design with pyridoxine (0, 0·25, or 0·5 mg l-1 ) and YAN (60 or 250 mg l-1 ) as variables, and (iii) 3 × 3 factorial design with thiamin (0, 0·2 or 0·5 mg l-1 ) and pyridoxine (0, 0·25 or 0·5 mg l-1 ) as variables in media containing 60 mg l-1 YAN. Although the progress of fermentations was affected by thiamin or pyridoxine, YAN had a larger impact than either vitamin. H2 S production was significantly lower with increasing amounts of thiamin in those fermentations under low YAN (60 mg l-1 ) while even lower amounts (<30 µg l-1 ) were produced under high YAN (250 mg l-1 ) with or without the vitamin. The highest amounts of H2 S were synthesized in those fermentations without any pyridoxine (>110 µg l-1 ), with the lowest production in media with pyridoxine and high YAN (<20 µg l-1 ). SIGNIFICANCE AND IMPACT OF THE STUDY: Concentrations of thiamin, pyridoxine and yeast assimilable nitrogen (YAN) influenced the synthesis of hydrogen sulphide (H2 S) by Saccharomyces cerevisiae in a synthetic grape juice medium. With a few exceptions, an increase in the concentration of either vitamin or YAN resulted in less H2 S released. This is the first report to demonstrate that both thiamin and pyridoxine along with YAN affected H2 S production, emphasizing the need to assess yeast nutrients to lower risks of off-odours during fermentation.

Small kernel2 Encodes a Glutaminase in Vitamin B6 Biosynthesis Essential for Maize Seed Development.

Vitamin B6, an essential cofactor for a range of biochemical reactions and a potent antioxidant, plays important roles in plant growth, development, and stress tolerance. Vitamin B6 deficiency causes embryo lethality in Arabidopsis (Arabidopsis thaliana), but the specific role of vitamin B6 biosynthesis in endosperm development has not been fully addressed, especially in monocot crops, where endosperm constitutes the major portion of the grain. Through molecular characterization of a small kernel2 (smk2) mutant in maize, we reveal that vitamin B6 has differential effects on embryogenesis and endosperm development in maize. The B6 vitamer pyridoxal 5'-phosphate (PLP) is drastically reduced in both the smk2 embryo and the endosperm. However, whereas embryogenesis of the smk2 mutant is arrested at the transition stage, endosperm formation is nearly normal. Cloning reveals that Smk2 encodes the glutaminase subunit of the PLP synthase complex involved in vitamin B6 biosynthesis de novo. Smk2 partially complements the Arabidopsis vitamin B6-deficient mutant pdx2.1 and Saccharomyces cerevisiae pyridoxine auxotrophic mutant MML21. Smk2 is constitutively expressed in the maize plant, including developing embryos. Analysis of B6 vitamers indicates that the endosperm accumulates a large amount of pyridoxamine 5'-phosphate (PMP). These results indicate that vitamin B6 is essential to embryogenesis but has a reduced role in endosperm development in maize. The vitamin B6 required for seed development is synthesized in the seed, and the endosperm accumulates PMP probably as a storage form of vitamin B6.

Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi.

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni-NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.

Effects of alanine:glyoxylate aminotransferase variants and pyridoxine sensitivity on oxalate metabolism in a cell-based cytotoxicity assay.

The hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) is caused by a functional deficiency of the liver-specific, peroxisomal, pyridoxal-phosphate-dependent enzyme, alanine:glyoxylate aminotransferase (AGT). One third of PH1 patients, particularly those expressing the p.[(Pro11Leu; Gly170Arg; Ile340Met)] mutant allele, respond clinically to pharmacological doses of pyridoxine. To gain further insight into the metabolic effects of AGT dysfunction in PH1 and the effect of pyridoxine, we established an "indirect" glycolate cytotoxicity assay using CHO cells expressing glycolate oxidase (GO) and various normal and mutant forms of AGT. In cells expressing GO the great majority of glycolate was converted to oxalate and glyoxylate, with the latter causing the greater decrease in cell survival. Co-expression of normal AGTs and some, but not all, mutant AGT variants partially counteracted this cytotoxicity and led to decreased synthesis of oxalate and glyoxylate. Increasing the extracellular pyridoxine up to 0.3µM led to an increased metabolic effectiveness of normal AGTs and the AGT-Gly170Arg variant. The increased survival seen with AGT-Gly170Arg was paralleled by a 40% decrease in oxalate and glyoxylate levels. These data support the suggestion that the effectiveness of pharmacological doses of pyridoxine results from an improved metabolic effectiveness of AGT; that is the increased rate of transamination of glyoxylate to glycine. The indirect glycolate toxicity assay used in the present study has potential to be used in cell-based drug screening protocols to identify chemotherapeutics that might enhance or decrease the activity and metabolic effectiveness of AGT and GO, respectively, and be useful in the treatment of PH1.

Methylmalonate-semialdehyde dehydrogenase mediated metabolite homeostasis essentially regulate conidiation, polarized germination and pathogenesis in Magnaporthe oryzae.

Plants generate multitude of aldehydes under abiotic and biotic stress conditions. Ample demonstrations have shown that rice-derived aldehydes enhance the resistance of rice against the rice-blast fungus Magnaporthe oryzae. However, how the fungal pathogen nullifies the inhibitory effects of host aldehydes to establish compatible interaction remains unknown. Here we identified and evaluated the in vivo transcriptional activities of M. oryzae aldehyde dehydrogenase (ALDH) genes. Transcriptional analysis of M. oryzae ALDH genes revealed that the acetylating enzyme Methylmalonate-Semialdehyde Dehydrogenase (MoMsdh/MoMmsdh) elevated activities during host invasion and colonization of the fungus. We further examined the pathophysiological importance of MoMSDH by deploying integrated functional genetics, and biochemical approaches. MoMSDH deletion mutant ΔMomsdh exhibited germination defect, hyper-branching of germ tube and failed to form appressoria on hydrophobic and hydrophilic surface. The MoMSDH disruption caused accumulation of small branch-chain amino acids, pyridoxine and AMP/cAMP in the ΔMomsdh mutant and altered Spitzenkörper organization in the conidia. We concluded that MoMSDH contribute significantly to the pathogenesis of M. oryzae by regulating the mobilization of Spitzenkörper during germ tube morphogenesis, appressoria formation by acting as metabolic switch regulating small branch-chain amino acids, inositol, pyridoxine and AMP/cAMP homeostasis.