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.

Pyridoxal 5'-Phosphate Biosynthesis by Pyridox-(am)-ine 5'-Phosphate Oxidase: Species-Specific Features.

Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.

Neopterin, kynurenine metabolites, and indexes related to vitamin B6 are associated with post-stroke cognitive impairment: The Nor-COAST study.

BACKGROUND AND AIMS: We have previously shown that systemic inflammation was associated with post-stroke cognitive impairment (PSCI). Because neopterin, kynurenine pathway (KP) metabolites, and B6 vitamers are linked to inflammation, in our study we investigated whether those biomarkers were associated with PSCI. MATERIAL AND METHODS: The Norwegian Cognitive Impairment After Stroke study is a prospective multicenter cohort study of patients with acute stroke recruited from May 2015 through March 2017. Plasma samples of 422 participants (59 % male) with ischemic stroke from the index hospital stay and 3 months post-stroke were available for analyses of neopterin, KP metabolites, and B6 vitamers using liquid chromatography-tandem mass spectrometry. Mixed linear regression analyses adjusted for age, sex, and creatinine, were used to assess whether there were associations between those biomarkers and cognitive outcomes, measured by the Montreal Cognitive Assessment scale (MoCA) at 3-, 18-, and 36-month follow-up. RESULTS: Participants had a mean (SD) age of 72 (12) years, with a mean (SD) National Institutes of HealthStroke Scale score of 2.7 (3.6) at Day 1. Higher baseline values of quinolinic acid, PAr (i.e., an inflammatory marker based on vitamin B6 metabolites), and HKr (i.e., a marker of functional vitamin B6 status based on selected KP metabolites) were associated with lower MoCA score at 3, 18, and 36 months post-stroke (p < 0.01). Higher baseline concentrations of neopterin and 3-hydroxykynurenine were associated with lower MoCA scores at 18 and 36 months, and higher concentrations of xanthurenic acid were associated with higher MoCA score at 36 months (p < 0.01). At 3 months post-stroke, higher concentrations of neopterin and lower values of pyridoxal 5́-phosphate were associated with lower MoCA scores at 18- and 36-month follow-up, while lower concentrations of picolinic acid were associated with a lower MoCA score at 36 months (p < 0.01). CONCLUSION: Biomarkers and metabolites of systemic inflammation, including biomarkers of cellular immune activation, indexes of vitamin B6 homeostasis, and several neuroactive metabolites of the KP pathway, were associated with PSCI. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02650531.

Discovery and characterization of natural product luteolin as an effective inhibitor of human pyridoxal kinase.

Pyridoxal kinase (PDXK) is an essential enzyme in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6, which plays a pivotal role in maintaining the enzyme activity necessary for cell metabolism. Thus, PDXK has garnered attention as a potential target for metabolism regulation and tumor therapy. Despite this interest, existing PDXK inhibitors have faced limitations, including weak suppressive activity, unclear mechanisms of action, and associated toxic side effects. In this study, we present the discovery of a novel PDXK inhibitor, luteolin, through a high-throughput screening approach based on enzyme activity. Luteolin, a natural product, exhibits micromolar-level affinity for PDXK and effectively inhibits the enzyme's activity in vitro. Our crystal structures reveal that luteolin occupies the ATP binding pocket through hydrophobic interactions and a weak hydrogen bonding pattern, displaying reversible characteristics as confirmed by biochemical assays. Moreover, luteolin disrupts vitamin B6 metabolism by targeting PDXK, thereby inhibiting the proliferation of leukemia cells. This research introduces a novel screening method for identifying high-affinity and potent PDXK inhibitors and sheds light on clarification of the structural mechanism of PDXK-luteolin for subsequent structure optimization of inhibitors.

Green Pea (Pisum sativum L.) Hull Polyphenol Extract Alleviates NAFLD through VB6/TLR4/NF-κB and PPAR Pathways.

Green pea hull is a processing byproduct of green pea and rich in polyphenols. Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disease characterized by accumulation of lipids in the liver for which there are no effective treatment strategies. Here, a mouse model of NAFLD induced by a DSS+high-fat diet (HFD) was established to investigate the effect of green pea hull polyphenol extract (EGPH). The results show that EGPH relief of NAFLD was a combined effect, including reducing hepatic fat accumulation, improving antioxidant activity and blood lipid metabolism, and maintaining glucose homeostasis. Increased intestinal permeability aggravated NAFLD. Combined metabolomics and transcriptomic analysis showed that vitamin B6 is the key target substance for EGPH to alleviate NAFLD, and it may be the intestinal flora metabolite. After EGPH intervention, the level of vitamin B6 in mice was significantly increased, and more than 60% in the blood enters the liver, which activated or inhibited PPAR and TLR4/NF-κB signaling pathways to relieve NAFLD. Our research could be a win-win for expanding the use of green pea hull and the search for NAFLD prophylactic drugs.

Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein.

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.

On pathways and blind alleys-The importance of biomarkers in vitamin B6 -dependent epilepsies.

Over the past two decades, the field of vitamin B6 -dependent epilepsies has evolved by the recognition of a growing number of gene defects (ALDH7A1, PNPO, ALPL, ALDH4A1, PLPBP as well as defects of the glycosylphosphatidylinositol anchor proteins) that all lead to reduced availability of pyridoxal 5'-phosphate, an important cofactor in neurotransmitter and amino acid metabolism. In addition, positive pyridoxine response has been observed in other monogenic defects such as MOCS2 deficiency or KCNQ2 and there may be more defects to be discovered. Most entities lead to neonatal onset pharmaco-resistant myoclonic seizures or even status epilepticus and pose an emergency to the treating physician. Research has unraveled specific biomarkers for several of these entities (PNPO deficiency, ALDH7A1 deficiency, ALDH4A1 deficiency, ALPL deficiency causing congenital hypophosphatasia and glycosylphosphatidylinositol anchoring defects with hyperphosphatasia), that can be detected in plasma or urine, while there is no biomarker to test for PLPHP deficiency. Secondary elevation of glycine or lactate was recognized as diagnostic pitfall. An algorithm for a standardized trial with vitamin B6 should be in place in every newborn unit in order not to miss these well-treatable inborn errors of metabolism. The Komrower lecture of 2022 provided me with the opportunity to tell the story about the conundrums of research into vitamin B6 -dependent epilepsies that kept some surprises and many novel insights into pathomechanisms of vitamin metabolism. Every single step had benefits for the patients and families that we care for and advocates for a close collaboration of clinician scientists with basic research.

Fusarium-produced vitamin B6 promotes the evasion of soybean resistance by Phytophthora sojae.

Plants can be infected by multiple pathogens concurrently in natural systems. However, pathogen-pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae-soybean-Fusarium combinations, more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen co-inoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1 (Fpp1), encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B6 (VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of disease-resistant crop varieties and provide new strategies to control soybean root rot.

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation.

Interleukin-33 (IL-33) is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis. The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation, but the mechanism is still unclear. Here, we found that healthy individuals had higher phosphate-pyridoxal (PLP, an active form of vitamin B6) concentrations in the serum than asthma patients. Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation. In a mouse model of lung inflammation, we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33. A mechanistic study showed that in vivo, pyridoxal (PL) needed to be converted into PLP, which inhibited the type 2 response by regulating IL-33 stability. In mice heterozygous for pyridoxal kinase (PDXK), the conversion of PL to PLP was limited, and IL-33 levels were increased in the lungs, aggravating type 2 inflammation. Furthermore, we found that the mouse double minute 2 homolog (MDM2) protein, an E3 ubiquitin-protein ligase, could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells. PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway. In addition, inhalation of PLP alleviated asthma-related effects in mouse models. In summary, our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response, which might help develop a potential preventive and therapeutic agent for allergy-related diseases.

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).

Characterization of a Potential Probiotic Lactiplantibacillus plantarum LRCC5310 by Comparative Genomic Analysis and its Vitamin B6 Production Ability.

Safety assessment and functional analysis of probiotic candidates are important for their industrial applications. Lactiplantibacillus plantarum is one of the most widely recognized probiotic strains. In this study we aimed to determine the functional genes of L. plantarum LRCC5310, isolated from kimchi, using next-generation, whole-genome sequencing analysis. Genes were annotated using the Rapid Annotations using Subsystems Technology (RAST) server and the National Center for Biotechnology Information (NCBI) pipelines to establish the strain's probiotic potential. Phylogenetic analysis of L. plantarum LRCC5310 and related strains showed that LRCC5310 belonged to L. plantarum. However, comparative analysis revealed genetic differences between L. plantarum strains. Carbon metabolic pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed that L. plantarum LRCC5310 is a homofermentative bacterium. Furthermore, gene annotation results indicated that the L. plantarum LRCC5310 genome encodes an almost complete vitamin B6 biosynthetic pathway. Among five L. plantarum strains, including L. plantarum ATCC 14917T, L. plantarum LRCC5310 detected the highest concentration of pyridoxal 5'-phosphate with 88.08 ± 0.67 nM in MRS broth. These results indicated that L. plantarum LRCC5310 could be used as a functional probiotic for vitamin B6 supplementation.

Transcriptional regulation mechanism of wheat varieties with different nitrogen use efficiencies in response to nitrogen deficiency stress.

BACKGROUND: As one of the microelements, nitrogen play essential roles in cereal production. Although the use of chemical fertilizers has significantly improved the yield of wheat, it has also caused increasingly adverse environmental pollution. Revealing the molecular mechanism manipulating wheat nitrogen use efficiency (NUE), and cultivating wheat germplasms with high nitrogen use efficiency has become important goals for wheat researchers. In this study, we investigated the physiological and transcriptional differences of three wheat cultivars with different NUE under low nitrogen stress. RESULTS: The results showed that, under low nitrogen conditions, the activities of nitrogen metabolism-related enzymes (GS, NR, GDH), antioxidant enzymes (SOD, POD, CAT) and soluble protein contents of ZM366 (high NUE cultivar) were higher than those of JD8 (low NUE cultivar). The hybrid cultivar of ZM366 and JD8 showed mid-parent or over-parent heterosis. Transcriptome analysis revealed that 'alanine, aspartate and glutamate metabolism', 'terpenoid backbone biosynthesis' and 'vitamin B6 metabolism' pathways play key roles in nitrogen use efficiency in wheat. The significant enhancement of the 'Calvin cycle' and 'photorespiration' in ZM366 contributed to its higher level of carbon metabolism under low nitrogen stress, which is an important attribute differs from the other two varieties. In addition, the activation of ABA signal transduction and biosynthesis pathways also helps to maintain NUE under low- nitrogen conditions. Moreover, bHLH transcription factors were also found to play a positive role in wheat NUE. CONCLUSIONS: In conclusion, these results enriched our knowledge of the mechanism of wheat NUE, and provided a theoretical basis for improving wheat NUE and breeding new cultivars.

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.

Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. In this work, we investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. Here, we found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, we identified the mitotic recombination as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

Pyridoxal Kinase of Disease-causing Human Parasites: Structural and Functional Insights to Understand its Role in Drug Discovery.

Human parasites cause several diseased conditions with high morbidity and mortality in a large section of the population residing in various geographical areas. Nearly three billion people suffer from either one or many parasitic infections globally, with almost one million deaths annually. In spite of extensive research and advancement in the medical field, no effective vaccine is available against prominent human parasitic diseases that necessitate identification of novel targets for designing specific inhibitors. Vitamin B6 is an important ubiquitous co-enzyme that participates in several biological processes and plays an important role in scavenging ROS (reactive oxygen species) along with providing resistance to oxidative stress. Moreover, the absence of the de novo vitamin B6 biosynthetic pathway in human parasites makes this pathway indispensable for the survival of these pathogens. Pyridoxal kinase (PdxK) is a crucial enzyme for vitamin B6 salvage pathway and participates in the process of vitamers B6 phosphorylation. Since the parasites are dependent on pyridoxal kinase for their survival and infectivity to the respective hosts, it is considered a promising candidate for drug discovery. The detailed structural analysis of PdxK from disease-causing parasites has provided insights into the catalytic mechanism of this enzyme as well as significant differences from their human counterpart. Simultaneously, structure-based studies have identified small lead molecules that can be exploited for drug discovery against protozoan parasites. The present review provides structural and functional highlights of pyridoxal kinase for its implication in developing novel and potent therapeutics to combat fatal parasitic diseases.

Is there any biological insight (or respite) for insects exposed to plastics? Measuring the impact on an insects central carbon metabolism when exposed to a plastic feed substrate.

Insects used to treat organic waste streams and produce valuable protein products are increasingly exposed to plastic contaminated source material assimilating plastic carbon into organic biomass, which is pervasive and hazardous to organisms. Our understanding of this increased insect-plastic interaction remains limited and needs urgent scientific attention if plastic biodegradation and production rates of quality protein are to be improved. Herein, we investigated the biochemical impact of various plastics using three insect models. Black Soldier Fly (BSF), Mealworm (MW), and Wax Moth (WM) larva were each exposed to a plastic substrate (PET, PE, PS, Expanded PE, PP, and PLA) as the primary carbon source for five days to explore any positive metabolic benefits in terms of insect performance and plastic degradation potential. Central carbon metabolism (CCM) metabolites were analyzed via a targeted tMRM liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) method. Unique expressed pathways were observed for each insect model. When reared on PET, BSF larvae were found to have an elevated pyrimidine metabolism, while the purine metabolism pathway was strongly expressed on other plastics. BSF also exhibited a downregulated Vitamin B6 metabolism across all plastics, indicating a likely gut-symbiont breakdown. The MW and WM model insects were metabolically more active on PLA and expanded foam plastics. Further, WM exhibited an elevation in Vitamin B6 metabolism. This data suggests a positive insect-specific interaction towards certain plastic types that warrants further investigation. It is anticipated that through deeper insight into the metabolic impact and benefits afforded from certain plastics, an insect biotransformation pipeline can be established that links fit-for-purpose insect models to individual plastic types that address our growing plastic waste issue.

Proteomic profiling for ovarian development and azadirachtin exposure in Spodoptera litura during metamorphosis from pupae to adults.

Azadirachtin is one of the most successful botanical pesticides in agricultural pest control. To build a repertoire of proteins and pathways in response to azadirachtin exposure during ovarian development, iTRAQ-based comparative proteomic was conducted. 1423 and 1686 proteins were identified as differentially accumulated proteins (DAPs) by comparing the protein abundance in adult ovary with that in pupal ovary under normal and azadirachtin exposure condition, respectively. Bioinformatics analysis indicated that pupae-to-adult transition requires proteins related to proteasome and branched chain amino acids (BCAAs) degradation for ovary development. Azadirachtin exposure strongly affected glycosylation-related pathway. And proteins related to vitamin B6 synthesis were necessary for ovary development under normal and AZA-exposure condition. RNAi assays confirmed the essential roles of DAPs related to glycosylation and vitamin B6 synthesis in moth growth and ovary development. The results enhance our understanding of the molecular regulatory network for ovary development and provide valuable resources for using AZA-responsive proteins to develop novel bio-rational insecticides.

Vitamin B6 Metabolism Determines T Cell Anti-Tumor Responses.

Targeting T cell metabolism is an established method of immunomodulation. Following activation, T cells engage distinct metabolic programs leading to the uptake and processing of nutrients that determine cell proliferation and differentiation. Redirection of T cell fate by modulation of these metabolic programs has been shown to boost or suppress immune responses in vitro and in vivo. Using publicly available T cell transcriptomic and proteomic datasets we identified vitamin B6-dependent transaminases as key metabolic enzymes driving T cell activation and differentiation. Inhibition of vitamin B6 metabolism using the pyridoxal 5'-phosphate (PLP) inhibitor, aminoxyacetic acid (AOA), suppresses CD8+ T cell proliferation and effector differentiation in a dose-dependent manner. We show that pyridoxal phosphate phosphatase (PDXP), a negative regulator of intracellular vitamin B6 levels, is under the control of the hypoxia-inducible transcription factor (HIF1), a central driver of T cell metabolism. Furthermore, by adoptive transfer of CD8 T cells into a C57BL/6 mouse melanoma model, we demonstrate the requirement for vitamin B6-dependent enzyme activity in mediating effective anti-tumor responses. Our findings show that vitamin B6 metabolism is required for CD8+ T cell proliferation and effector differentiation in vitro and in vivo. Targeting vitamin B6 metabolism may therefore serve as an immunodulatory strategy to improve anti-tumor immunotherapy.

Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes.

Pyridox(am)ine 5 ' -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 ' -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in Drosophila alleles, hWT , hR116Q , hD33V , and hR95H , in which the endogenous Drosophila PNPO was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that hR95H had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.

Genetic Analysis Using Vitamin B6 Antagonist 4-Deoxypyridoxine Uncovers a Connection between Pyridoxal 5'-Phosphate and Coenzyme A Metabolism in Salmonella enterica.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for organisms in all three domains of life. Despite the central role of PLP, many aspects of vitamin B6 metabolism, including its integration with other biological pathways, are not fully understood. In this study, we examined the metabolic perturbations caused by the vitamin B6 antagonist 4-deoxypyridoxine (dPN) in a ptsJ mutant of Salmonella enterica serovar Typhimurium LT2. Our data suggest that PdxK (pyridoxal/pyridoxine/pyridoxamine kinase [EC 2.7.1.35]) phosphorylates dPN to 4-deoxypyridoxine 5'-phosphate (dPNP), which in turn can compromise the de novo biosynthesis of PLP. The data are consistent with the hypothesis that accumulated dPNP inhibits GlyA (serine hydroxymethyltransferase [EC 2.1.2.1]) and/or GcvP (glycine decarboxylase [EC 1.4.4.2]), two PLP-dependent enzymes involved in the generation of one-carbon units. Our data suggest that this inhibition leads to reduced flux to coenzyme A (CoA) precursors and subsequently decreased synthesis of CoA and thiamine. This study uncovers a link between vitamin B6 metabolism and the biosynthesis of CoA and thiamine, highlighting the integration of biochemical pathways in microbes. IMPORTANCE PLP is a ubiquitous cofactor required by enzymes in diverse metabolic networks. The data presented here expand our understanding of the toxic effects of dPN, a vitamin B6 antagonist that is often used to mimic vitamin B6 deficiency and to study PLP-dependent enzyme kinetics. In addition to de novo PLP biosynthesis, we define a metabolic connection between vitamin B6 metabolism and synthesis of thiamine and CoA. This work provides a foundation for the use of dPN to study vitamin B6 metabolism in other organisms.