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The cysteine desulfurase SufS is a pyridoxal-5'-phosphate-dependent enzyme and is essential for the SUF system, which participates in iron-sulfur cluster biosynthesis. Inhibition of SufS in the SUF system by D-cycloserine (DCS) in Plasmodium falciparum apicoplast has recently been reported, indicating that SufS could be a target for malaria therapeutics. However, the mechanistic details underlying the inhibition of SufS by DCS have not yet been clarified. Moreover, inhibition of SufS by the other enantiomer, L-cycloserine (LCS), has not been investigated. Herein, we investigated the structure-based inhibition mechanisms of SufS by DCS and LCS using Bacillus subtilis SufS, whose catalytic mechanism has been well characterized in comparison to that of the P. falciparum SufS. Surprisingly, DCS- and LCS-mediated inhibitions of SufS occur via distinct mechanisms resulting in pyridoxamine-5'-phosphate (PMP) in DCS-mediated inhibition and PMP-3-hydroxyisoxazole adduct (PMP-isoxazole) in LCS-mediated inhibition. Biochemical and structural evaluation of SufS variants identified conserved His and Arg residues at the active site as the key determinants of the distinct inhibition mechanisms. The importance of structural elements involved in DCS and LCS-mediated inhibitions of SufS provides valuable insights for the structure-based design of new drugs targeting SufS. DATABASE: Structural data are available in PDB database under the accession numbers 6KFY, 7CEO, 7CEP, 7CEQ, 7CER, 7CES, 7CET, 7CEU, 7E6A, 7E6B, 7E6C, 7E6D, 7E6E, and 7E6F.

Abstract The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6. Key points • Known vitamin B6 routes have been genetically engineered. • Underground metabolism facilitates the emergence of novel vitamin B6 biosynthetic pathways. • These pathways may be suitable to engineer bacteria overproducing vitamin B6.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent K(m) of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

Oxidation of docosahexaenoate (DHA)-containing phospholipids in the cell plasma membrane leads to release of the α,β-unsaturated aldehyde 4-hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone which is capable of inducing retinal pigmented epithelial (RPE) cell dysfunction. Previously, HOHA lactone was shown to induce apoptosis and angiogenesis, and to activate the alternative complement pathway. RPE cells metabolize HOHA lactone through enzymatic conjugation with glutathione (GSH). Competing with this process is the adduction of HOHA lactone to protein lysyl residues generating 2-(ω-carboxyethyl)pyrrole (CEP) derivatives that have pathological relevance to age-related macular degeneration (AMD). We now find that HOHA lactone induces mitochondrial dysfunction. It decreases ATP levels, mitochondrial membrane potentials, enzymatic activities of mitochondrial complexes, depletes GSH and induces oxidative stress in RPE cells. The present study confirmed that pyridoxamine and other primary amines, which have been shown to scavenge γ-ketoaldehydes formed by carbohydrate or lipid peroxidation, are ineffective for scavenging the α,β-unsaturated aldehydes. Histidyl hydrazide (HH), that has both hydrazide and imidazole nucleophile functionalities, is an effective scavenger of HOHA lactone and it protects ARPE-19 cells against HOHA lactone-induced cytotoxicity. The HH α-amino group is not essential for this electrophile trapping activity. The Nα-acyl L-histidyl hydrazide derivatives with 2- to 7-carbon acyl groups with increasing lipophilicities are capable of maintaining the effectiveness of HH in protecting ARPE-19 cells against HOHA lactone toxicity, which potentially has therapeutic utility for treatment of age related eye diseases.

Defects of vitamin B6 metabolism are responsible for severe neurological disorders, such as pyridoxamine 5′-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5′-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B6 metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.

The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5'-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5'-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors.

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5′-phosphate was determined at 2.05 Å resolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.

International audience; Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder for which dietary interventions can be a useful treatment. In recent years, the low-FODMAP approach is gaining traction in this regard. The fermentation of these non-absorbed carbohydrates by the gut microbiota can generate toxic glycating metabolites, such as methylglyoxal. These metabolites can have harmful effects by their role in the generation of advanced glycation end products (AGEs), which activates Receptor for AGEs (AGER). Mast cells can be stimulated by AGEs and play a role in IBS. We have treated mice with lactose or fructo-oligosaccharides (FOS), with or without co-administration of pyridoxamine and investigated the colonic mucus barrier. We have found that an increased intake of lactose and fructo-oligosaccharides induces a dysregulation of the colonic mucus barrier, increasing mucus discharge in empty colon, while increasing variability and decreasing average thickness mucus layer covering the fecal pellet. Changes were correlated with increased mast cell counts, pointing to a role for the crosstalk between these and goblet cells. Additionally, AGE levels in colonic epithelium were increased by treatment with the selected fermentable carbohydrates. Observed effects were prevented by co-treatment with anti-glycation agent pyridoxamine, implicating glycation processes in the negative impact of fermentable carbohydrate ingestion. This study shows that excessive intake of fermentable carbohydrates can cause colonic mucus barrier dysregulation in mice, by a process that involves glycating agents and increased mucosal mast cell counts.

Chlorine is a widely used disinfectant and oxidant used for an array of municipal and industrial applications, including potable water, swimming pools, and cleaning of membranes. The most popular method to verify the concentration of free chlorine is the colorimetric method based on DPD (N, N-diethyl-p-phenylenediamine), which is fast and reasonably cheap, but DPD and its product are potentially toxic. Therefore, a novel, environmentally friendly colorimetric method for the quantification of residual chlorine based on the food additive pyridoxamine (4-(aminomethyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol) was investigated. Pyridoxamine is a B6 vitamin with an absorption maximum at 324 nm and fluorescence emission at 396 nm. Pyridoxamine reacts rapidly and selectively with free chlorine, resulting in a linear decrease both in absorbance and in emission, giving therefore calibration curves with a negative slope. The pyridoxamine method was successfully applied for the quantification of free chlorine from 0.2 to 250 mg/L. Using 1 cm cuvettes, the limit of quantification was 0.12 mg Cl2/L. The pyridoxamine and the DPD methods were applied to actual environmental samples, and the deviation between results was between 4% and 9%. While pyridoxamine does not react with chloramine, quantification of monochloramine was possible when iodide was added, but the reaction is unfavourably slow.

Detoxification of reactive carbonyl compounds (RCC) is crucial to sustain cellular activity to improve plant growth and development. Seedling growth is highly affected by accumulation of RCC under stress. We report non-enzymatic, enzymatic mechanisms of detoxification of RCC in the cucumber, tobacco and rice seedling systems exposed to glucose, NaCl, methyl viologen (MV) induced oxidative stress. The cucumber seedlings exposed to carbonyl stress had higher levels of malondialdehyde (MDA), protein carbonyls (PCs) and advanced glycation end-product N-carboxymethyl-lysine (AGE-CML) that negatively affected the seedling growth. The overexpression of enzyme encoding aldo-keto reductase-1 (AKR1) in tobacco and rice showed detoxification of RCC, MDA and methylglyoxal (MG) with improved seedling growth under glucose, NaCl and MV-induced oxidative stress. Further, small molecules like acetylsalicylic acid (ASA), aminoguanidine (AG), carnosine (Car), curcumin (Cur) and pyridoxamine (PM) showed detoxification of RCC non-enzymatically and rescued the cucumber seedling growth from glucose, NaCl and MV-stress. In autotrophically grown rice seedlings these molecules substantially improved seedling growth under MV-induced oxidative stress. Seedlings treated with the small molecules sustained higher guaiacol peroxidase (GPX) enzyme activity signifying the role of small molecules in reducing carbonyl stress-induced protein inactivation and AGE-CML protein modifications. The results showed that besides enzymatic detoxification of RCC, the small molecules also could reduce cytotoxic effect of RCC under stress. The study demonstrates that small molecules are attractive compounds to improve the seedling growth under stress conditions.

The antioxidant and antiglycation activity of the water-soluble heterocyclic antioxidant 6‑hydroxy-2-aminobenzothiazole N-acetylcysteinate (HABTI ACC) has been studied. The high antiradical activity of this substance has been found by the chemiluminescence method. HABTI ACC has been found to show the high antioxidant activity in the peroxidation of photoreceptor cells induced by ultraviolet radiation, ferrous ions, and visible light in the presence of age pigment lipofuscin or its A2E fluorophore. HABTI ACC effectively inhibits the fructosylation of serum albumin and proteins of photoreceptor cells. The HABTI ACC inhibitory ability in the process of fructose albumin modification is comparable with the aminoguanidine and pyridoxamine activity, the known antiglycation compounds, and is much higher than that of mexidol. It is suggested that the use of this compound is promising in pharmacological practice for the prevention and treatment of metabolic syndrome.

Pyridoxine and its derivatives, pyridoxamine and pyridoxal, are the three main forms of vitamin B6, which play exceptionally important biological roles in living organisms. The active endogenous metabolites of these molecules, pyridoxal phosphate and pyridoxamine phosphate, are the most important coenzymes involved in a wide range of biochemical reactions necessary for cell activity, due to which pyridoxine and its derivatives are regarded as biologically privileged molecules. Taking into account also the wide possibilities for chemical modification of the pyridoxine structure, it is only natural for medicinal chemists to explore them in the design of novel drugs. This review summarizes the data on the main pharmacologically significant pyridoxine derivatives (including pyridoxamine and pyridoxal derivatives) reported in modern scientifi c and patent sources. Methods for their synthesis, key pharmacological properties, and medicinal chemistry concepts underlying the design of the developing physiologically active compounds are presented. The promising directions for future development of chemistry of physiologically active pyridoxine derivatives are also discussed.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Recent studies have shown that carbonyl stress is a causative factor of schizophrenia, categorized as carbonyl stress-related schizophrenia (CS-SCZ). However, the correlation between carbonyl stress and the pathogenesis of this disease is not well established. In this study, glyoxalase 1(Glo1)-knockout and vitamin B6-deficient mice (KO/VB6 (-) mice), which are susceptible to methylglyoxal (MGO)-induced oxidative damages, were used as a CS-SCZ model to analyze MGO-modified protein and the carbonyl stress status in the brain. A comparison between Wild/VB6(+) mice and KO/VB6(−) mice for accumulated carbonyl proteins levels, with several advanced glycation end products (AGEs) in the brain, revealed that carbonyl protein levels with the Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) moiety were significantly increased in the hippocampus, prefrontal cortex, striatum, cerebral cortex, and brainstem regions of the brain in KO/VB6(−) mice. Moreover, two-dimensional electrophoresis and Liquid chromatography-tandem mass spectrometry analysis showed MG-H1-modified arginine residues in mitochondrial creatine kinase, beta-adrenergic receptor kinase 1, and T-complex protein in the hippocampus region of KO/VB6(−) mice, but not in Wild/VB6(+) mice. In particular, MG-H1 modification of mitochondrial creatine kinase was quite notable. These results suggest that further studies focusing on MG-H1-modified and accumulated proteins in the hippocampus may reveal the onset mechanism of CS-SCZ induced by MGO-induced oxidative damages.

The thermodynamic stability of 11 complexes of Cu(II) and 26 complexes of Fe(III) is studied, comprising the ligands pyridoxamine (PM), ascorbic acid (ASC), and a model Amadori compound (AMD). In addition, the secondary antioxidant activity of PM is analyzed when chelating both Cu(II) and Fe(III), relative to the rate constant of the first step of the Haber-Weiss cycle, in the presence of the superoxide radical anion (O2•−) or ascorbate (ASC−). Calculations are performed at the M05(SMD)/6-311+G(d,p) level of theory. The aqueous environment is modeled by making use of the SMD solvation method in all calculations. This level of theory accurately reproduces the experimental data available. When put in perspective with the stability of various complexes of aminoguanidine (AG) (which we have previously studied), the following stability trends can be found for the Cu(II) and Fe(III) complexes, respectively: ASC &lt, AG &lt, AMD &lt, PM and AG &lt, ASC &lt, PM. The most stable complex of Cu(II) with PM (with two bidentate ligands) presents a ΔGf0 value of −35.8 kcal/mol, whereas the Fe(III) complex with the highest stability (with three bidentate ligands) possesses a ΔGf0 of −58.9 kcal/mol. These complexes can significantly reduce the rate constant of the first step of the Haber-Weiss cycle with both O2•− and ASC−. In the case of the copper-containing reaction, the rates are reduced up to 9.70 × 103 and 4.09 × 1013 times, respectively. With iron, the rates become 1.78 × 103 and 4.45 × 1015 times smaller, respectively. Thus, PM presents significant secondary antioxidant activity since it is able to inhibit the production of ·OH radicals. This work concludes a series of studies on secondary antioxidant activity and allows potentially new glycation inhibitors to be investigated and compared relative to both PM and AG.

High plasma levels of the sphingolipid intermediates ceramide (Cer) and sphingosine-1-phosphate (S1P) are suggested to be involved in the development of insulin resistance (IR). Recent evidence indicates that advanced glycation endproducts (AGEs) can alter the sphingolipids metabolism equilibrium. Since enzymes responsible for sphingolipid rheostat maintenance are highly expressed in liver, we thus investigated whether AGEs accumulation can affect hepatic sphingolipids metabolism in insulin resistant mice. Two different models of IR were examined: genetically diabetic LeptrDb-/- (DbDb) and diet-induced insulin resistant C57Bl/6J mice fed a 60% trans-fat diet (HFD). In addition, a group of HFD mice was supplemented with the anti-AGEs compound pyridoxamine. AGEs were evaluated in the liver by western blotting. Cer and S1P were measured by UHPLC-MS/MS. The expression of RAGE and of enzymes involved in sphingolipid metabolism were assessed by RT-PCR and western blotting. HepG2 cells were used to study the effect of the major AGE Ne-(carboxymethyl)lysine (CML)-albumin on sphingolipid metabolism and the role of the receptor of AGEs (RAGE). High levels of AGEs and RAGE were detected in the liver of both DbDb and HFD mice in comparison to controls. The expression of enzymes of sphingolipid metabolism was altered in both models, accompanied by increased levels of Cer and S1P. Specifically, ceramide synthase 5 and sphingosine kinase 1 were increased, while neutral ceramidase was reduced. Pyridoxamine supplementation to HFD mice diminished hepatic AGEs and prevented alterations of sphingolipid metabolism and the development of IR. CML administration to HepG2 cells evoked alterations similar to those observed in vivo, that were in part mediated by the binding to RAGE. The present study shows a direct involvement of AGEs in alterations of sphingolipid metabolism associated to the development of IR. The modulation of sphingolipids metabolism through the prevention of AGEs accumulation by pyridoxamine may reduce the development of IR.

Vitamin B6 is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP. Pyridoxal 5'-phosphate is a coenzyme in a variety of enzyme reactions concerning transformations of amino and amino acid compounds. This review summarizes all known and putative PLP-binding proteins found in the Escherichia coli MG1655 proteome. PLP can have toxic effects since it contains a very reactive aldehyde group at its 4' position that easily forms aldimines with primary and secondary amines and reacts with thiols. Most PLP is bound either to the enzymes that use it as a cofactor or to PLP carrier proteins, protected from the cellular environment but at the same time readily transferable to PLP-dependent apoenzymes. E. coli and its relatives synthesize PLP through the seven-step deoxyxylulose-5-phosphate (DXP)-dependent pathway. Other bacteria synthesize PLP in a single step, through a so-called DXP-independent pathway. Although the DXP-dependent pathway was the first to be revealed, the discovery of the widespread DXP-independent pathway determined a decline of interest in E. coli vitamin B6 metabolism. In E. coli, as in most organisms, PLP can also be obtained from PL, PN, and PM, imported from the environment or recycled from protein turnover, via a salvage pathway. Our review deals with all aspects of vitamin B6 metabolism in E. coli, from transcriptional to posttranslational regulation. A critical interpretation of results is presented, in particular, concerning the most obscure aspects of PLP homeostasis and delivery to PLP-dependent enzymes.

Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6 and an essential cofactor in all organisms. In Escherichia coli, PLP is synthesized via the deoxyxylulose 5-phosphate (DXP)-dependent pathway that includes seven enzymatic steps and generates pyridoxine 5′-phosphate as an intermediate. Additionally, E. coli is able to salvage pyridoxal, pyridoxine, and pyridoxamine B6 vitamers to produce PLP using kinases PdxK/PdxY and pyridox(am)ine phosphate oxidase (PdxH). We found that E. coli strains blocked in PLP synthesis prior to the formation of pyridoxine 5′-phosphate (PNP) required significantly less exogenous pyridoxal (PL) than strains lacking pdxH and identified the conversion of PL to pyridoxine (PN) during cultivation to be the cause. Our data showed that PdxI, shown to have PL reductase activity in vitro, was required for the efficient salvage of PL in E. coli. The pdxI+ E. coli strains converted exogenous PL to PN during growth, while pdxI mutants did not. In total, the data herein demonstrated that PdxI is a critical enzyme in the salvage of PL by E. coli. IMPORTANCE The biosynthetic pathway of pyridoxal 5′-phosphate (PLP) has extensively been studied in Escherichia coli, yet limited information is available about the vitamin B6 salvage pathway. We show that the protein PdxI (YdbC) is the primary pyridoxal (PL) reductase in E. coli and is involved in the salvage of PL. The orthologs of PdxI occur in a wide range of bacteria and plants, suggesting that PL reductase in the B6 salvage pathway is more widely distributed than previously expected.

Artifacts due to metabolite extraction, derivatization, and detection techniques can result in aberrant observations that are not accurate representations of actual cell metabolism. Here, we show that α-ketoglutarate (α-KG) is reductively aminated to glutamate in methanol:water metabolite extracts, which introduces an artifact into metabolomics studies. We also identify pyridoxamine and urea as amine donors for α-KG to produce glutamate in methanol:water buffer in vitro, and we demonstrate that the addition of ninhydrin to the methanol:water buffer suppresses the reductive amination of α-KG to glutamate in vitro and in metabolite extracts. Finally, we calculate that glutamate levels have been overestimated by 10-50%, depending on cell line, due to α-KG reductive amination. These findings suggest that precautions to account for α-KG reductive amination should be taken for the accurate quantification of glutamate in metabolomics studies.

New uracil and thymine derivatives, N1-,N3- and N1,N3-(RO-benzoyl)-(1H,3H)-pyrimidine- 2,4-diones, were synthesized (RO- is hydroxy, acetoxy- or methoxy-group). The compounds were studied in a complex of in vitro tests for the ability to inhibit the development of long-term complications of diabetes. Their ability to cleave cross-links of proteins has been evaluated. The most significant ways of pharmacological correction of thrombosis, angio-, nephro-, encephalo-, and cardiopathy, antiglycation, chelating, and antiplatelet activities, have been established. The most active compound in terms of antiplatelet action, N1- hydroxybenzoyluracil, exceeded acetylsalicylic acid by ~44%. In terms of their ability to chelate copper (II) cations, all compounds (with the exception of 1,3-bis(3-hydroxybenzoyl)-(1H,3H)-pyrimidine-2,4-dione that was not not studied in this test) showed the activity, whose IC50 fell in the range between that for pioglitazone (44.1 μM) and pyridoxamine (136.7 μM) comparison drugs. The best antiglycation effect at the 1 mM concentration was observed for N1,N3-bismethoxy- and N1,N3-bisacetoxybenzoyl derivatives of thymine. The maximum activity to cleave cross-links of proteins (C = 1 mM), comparable to that of alagebrium, was established for 1,3-bis(4-methoxybenzoyl)uracil, for which also high rates of other estimated activities were noted. Thus, the N1-,N3- and N1,N3-(RO-benzoyl) derivatives of uracil and thymine are promising basiсs for creating drugs that suppress the development of long-term complications of diabetes.

B6 vitamers are important natural compounds for human life, principally for amino acid metabolism, and have been reported as potent scavenger molecules of both endogenous and exogenous reactive oxygen species (ROS). However, under UV-light excitation from UVC (200–280 nm) to UVA (315–400 nm), B6 vitamers act as endogenous photosensitizers that promote both cellular photodamage and enzyme inactivation. This occurs through classical photochemical mechanisms mediated by ROS, toxic photoproducts, and the formation of adducts with active protein sites. This minireview aimed to present and discuss the dual roles of B6 vitamers as generators and scavengers of singlet oxygen (an important reactive species in photodynamic processes) and various other ROS. We also examine the basic photophysical, photochemical, and protolytic equilibria principles of B6 vitamers and their role as photodamaging and photoprotecting compounds in UV-light mediated photobiological processes.

Aim The aim of this clinical trial was to obtain proof of concept for high-dose pyridoxamine as a novel treatment for schizophrenia with enhanced carbonyl stress. Methods Ten Japanese schizophrenia patients with high plasma pentosidine, which is a representative biomarker of enhanced carbonyl stress, were recruited in a 24-week, open trial in which high-dose pyridoxamine (ranging from 1200 to 2400 mg/day) was administered using a conventional antipsychotic regimen. Main outcomes were the total change in Positive and Negative Syndrome Scale score and the Brief Psychiatric Rating Scale score from baseline to end of treatment at week 24 (or at withdrawal). Results Decreased plasma pentosidine levels were observed in eight patients. Two patients showed marked improvement in their psychological symptoms. A patient who harbors a frameshift mutation in the Glyoxalase 1 gene also showed considerable reduction in psychosis accompanied with a moderate decrease in plasma pentosidine levels. A reduction of greater than 20% in the assessment scale of drug-induced Parkinsonism occurred in four patients. Although there was no severe suicide-related ideation or behavior, Wernicke's encephalopathy-like adverse drug reactions occurred in two patients and were completely suppressed by thiamine supplementation. Conclusion High-dose pyridoxamine add-on treatment was, in part, effective for a subpopulation of schizophrenia patients with enhanced carbonyl stress. Further randomized, placebo-controlled trials with careful monitoring will be required to validate the efficacy of high-dose pyridoxamine for these patients.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target. (Less)

In diabetics, formation of advanced glycation end (AGE) products has been closely associated with cardiovascular complications. In order to mitigate the injury caused by AGEs, it is critical to und...

UVA-driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3-hydroxypyridine-derived chromophores including B6 -vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA-induced photooxidative stress in human skin cells. Here, we report that the B6 -vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA-induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ-H2AX (Ser139)], comet analysis indicated the formation of Fpg-sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA-driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B6 -vitamers.

Continous renal replacement therapy (CRRT) is particularly recommended for septic shock patients in intensive care units. The CRRT technique used most frequently is high volume continuous veno-venous haemofiltration. It provides a high rate of clearance of uremic toxins and inflammatory cytokines. However, it should also be taken into account that substances important for homeostasis may be concurrently unintentionally removed. Accordingly, water-soluble vitamins can be removed during continuous renal replacement therapy, and the estimate of the loss is critical to ensure appropriate supplementation. The aim of this work was to develop a simple methodology for a purification step prior to the LC-MS/MS determination of water-soluble vitamins in ultrafiltrate samples. For this purpose, two types of resin and a mix of resins were used as sorbents for the purification step. Moreover, parameters such as the amount of resin and the extraction time were optimized. The LC-MS/MS method was developed and validated for final determination of 11 vitamins. The results demonstrated the high purification capability of DEAE Sephadex resin with recoveries between 65 and 101% for water-soluble vitamins from ultrafiltrate samples. An optimized method was applied to assess the loss of B-group vitamins in patients after 24h of renal replacement therapy. The loss of vitamins B2, B6 pyridoxamine, B6 pyridoxal, B7, B1, and B5 in ultrafiltrates was similar in all patients. In the native ultrafiltrates, vitamins B6 pyridoxine, B9 and B12 were not detected.

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.

A new type of chiral pyridoxamines bearing an adjacent chiral stereocenter has been developed via multi-step synthesis. The pyridoxamines displayed catalytic activity in asymmetric transamination of α-keto acids to give a variety of optically active amino acids in 27–78% yields with 34–62% ee’s under very mild conditions. This work provides a synthetic strategy to construct new chiral pyridoxamines using bromopyridine 7 as a key synthon and also represents an early example of the applications of chiral pyridoxamines in asymmetric catalysis.

Pyridoxamine, a reactive carbonyl (RCO) scavenger, can ameliorate peritoneal deterioration in uremic peritoneal dialysis (PD) rats when given via dialysate. We examined the effects of scavenging circulating RCOs by oral pyridoxamine. Rats underwent nephrectomy and 3 weeks of twice daily PD either alone or with once daily oral pyridoxamine. PD solution was supplemented with methylglyoxal, a major glucose-derived RCO, to quench intraperitoneal pyridoxamine. Oral pyridoxamine achieved comparable blood and dialysate pyridoxamine concentrations, suppressed pentosidine accumulation in the blood but not in the mesenterium or dialysate, and reduced the increases in small solute transport and mesenteric vessel densities, with no effects on submesothelial matrix layer thickening or serum creatinine. Thus, reducing circulating RCOs by giving oral pyridoxamine with PD provides limited peritoneal protection. However, orally given pyridoxamine efficiently reaches the peritoneal cavity and would eliminate intraperitoneal RCOs. Oral pyridoxamine is more clinically favorable and may be as protective as intraperitoneal administration.

Enzymatic transamination is catalyzed by pyridoxal/pyridoxamine, and it involves remarkable cooperative catalysis of a Lys residue in the transaminase. Inspired by transaminases, we developed a class of axially chiral pyridoxamines 11 bearing a lateral amine arm. The pyridoxamines exhibited high catalytic activity and excellent enantioselectivity in asymmetric transamination of α-keto acids, to give various α-amino acids in 67-99% yields with 83-94% ee's. The lateral amine arm likely participates in cooperative catalysis as the Lys residue does in biological transamination and has an important impact on the transamination in terms of activity and enantioselectivity.

The pyridoxal 5-phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ-aminobutyric acid aminotransferase (GABA-AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA-AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA-AT. This is a consequence of a pKa shift triggered by a strong charge-charge interaction with an ionic "diad" formed by Asp298 and His190 that would help the activation of the first half-reaction of the catalytic mechanism in GABA-AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π-π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half-reaction in GABA-AT: the regeneration of PLP-bound GABA-AT (i.e., the holoenzyme). Proteins 2016; 84:875-891. © 2016 Wiley Periodicals, Inc.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for more than 140 enzymes. This coenzyme plays a pivotal role in catalysis of various enzymatic reactions that are critical for the survival of organisms. Entamoeba histolytica depends on the uptake of pyridoxal (PL), a B6 vitamer from the external environment which is then phosphorylated by pyridoxal kinase (EhPLK) to form PLP via the salvage pathway. E. histolytica cannot synthesise vitamin B6de-novo, and also lacks pyridoxine 5'-phosphate oxidase, a salvage pathway enzyme required to produce PLP from pyridoxine phosphate (PNP) and pyridoxamine phosphate (PMP). Analysing the importance of PLK in E. histolytica, we have determined the high-resolution crystal structures of the dimeric pyridoxal kinase in apo, ADP-bound, and PLP-bound states. These structures provided a snapshot of the transition state and help in understanding the reaction mechanism in greater detail. The EhPLK structure significantly differed from the human homologue at its PLP binding site, and the phylogenetic study also revealed its divergence from human PLK. Further, gene regulation of EhPLK using sense and antisense RNA showed that any change in optimal level is harmful to the pathogen. Biochemical and in vivo studies unveiled EhPLK to be essential for this pathogen, while the molecular differences with human PLK structure can be exploited for the structure-guided design of EhPLK inhibitors.

Pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK) cooperate to produce pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PDXK phosphorylates pyridoxine, pyridoxamine, and pyridoxal by producing PNP, PMP, and PLP, whereas PNPO oxidizes PNP, PMP, into PLP. We previously demonstrated that PDXK depletion in Drosophila and human cells impacts on glucose metabolism and DNA integrity. Here we characterized sgll, the Drosophila ortholog of PNPO gene, showing that its silencing by RNA interference elicits chromosome aberrations (CABs) in brains and induces diabetic hallmarks such as hyperglycemia and small body size. We showed that in sgllRNAi neuroblasts CABs are largely produced by the genotoxic effect of the advanced glycation end products triggered by high glucose. As in sgllRNAi cells, part of PLP is still produced by PDXK activity, these data suggest that PLP dosage need to be tightly regulated to guarantee glucose homeostasis and DNA integrity.

Hyperglycaemia is considered to be a driving factor for advanced glycated end products (AGEs). Inhibiting the process of glycation play an important role in reducing the diabetes related complications. We have explored the glucose mediated glycation and antiglycation activity of pyridoxamine using human serum albumin (HSA). Protein was incubated with glucose for 28 days at physiological temperature to achieve glycation. Antiglycation activity was assessed by the estimation of carbonyl content, free lysine and AGE specific fluorescence. Molecular docking was used to study the interaction of pyridoxamine with HSA and to get a detailed understanding of binding sites and binding energy. Glycation was reduced by pyridoxamine to commendable levels which was evident by the quantification of free lysine and carbonyl content. Pyridoxamine treatment also prevented the loss in secondary structure induced by glycation. It has also emerged as the quencher of reactive oxygen species which lead to the protection of DNA from oxidative damage. Pyridoxamine was found to be located at subdomain IIA of HSA with binding energy of −5.6 kcal/mol. These results are high points in the antiglycation activity of pyridoxamine. Its antioxidant nature and antiglycation activity are proof of its potential in preventing disease progression in diabetes.

Pyridoxamine, which is a form of vitamin B6, is a promising candidate for a prophylactic and/or remedy for diabetic complications. Pyridoxamine is chemically synthesized by an oxidative method in manufacturing. However, pyridoxamine production by bioconversion, which is generally preferable for environmental and energetic aspects, has been little investigated. Therefore, I aimed to produce pyridoxamine from pyridoxine, which is a readily and economically available starting material, by bioconversion using a Rhodococcus expression system. I found in the bioconversion of pyridoxine to pyridoxal, approximately 450 mM pyridoxal was produced from 500 mM pyridoxine using recombinant Rhodococcus erythropolis expressing the pyridoxine 4-oxidase gene derived from Mesorhizobium loti. Next, in the bioconversion of pyridoxal to pyridoxamine using recombinant R. erythropolis expressing the pyridoxamine-pyruvate aminotransferase gene derived from M. loti, the bioconversion rate was approximately 80% under the same conditions as pyridoxal production. Finally, in the bioconversion of pyridoxine to pyridoxamine through pyridoxal using recombinant R. erythropolis coexpressing the genes for pyridoxine 4-oxidase and pyridoxamine-pyruvate aminotransferase, the bioconversion rate was approximately 75%. Based on these findings, pyridoxamine production by bioconversion using a Rhodococcus expression system may be of interest for future industrial applications.

Pyridoxamine (PM) is a promising drug candidate for treating various chronic conditions/diseases in which oxidative stress and carbonyl compounds are important factors affecting pathogenicity. These abilities of PM are mainly attributed to its inhibition of advanced glycation and lipoxidation end product formation, by scavenging reactive carbonyl species. PM might therefore prevent protein damage from lipid hydroperoxide-derived aldehydes such as 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) by trapping them. It was previously reported that PM reacts with ONE to produce pyrrolo-1,3-oxazine (PO8) through the formation of pyrido-1,3-oxazine (PO1/PO2). In this study, we found that ONE and HNE yield an identical product containing a pyrrole ring (PO7, PH2) upon reaction with PM. The structure of PO7/PH2 was shown by LC-MS and NMR analyses to be 1-(2-hydroxy-6-hydroxymethyl-3-methylpyridin-4-ylmethyl)-2-pentylpyrrole. PO1, PO7/PH2, and PO8 were the main stable PM-ONE/HNE adducts. In the incubation of human serum albumin (HSA) with ONE or HNE, Lys residues provided the most favorable modification sites for both aldehydes, and the number of HNE-modified sites was higher than that of ONE-modified sites. When HSA was allowed to react with a linoleic acid hydroperoxide in the presence of ascorbic acid, ONE modified more residues (10 Lys, 3 His, 2 Arg) than did HNE (8 His, 2 Lys), indicating the relative reactivity of aldehydes towards amino acid residues. Upon treatment with increasing concentrations of PM, the concentrations of ONE-modified HSA peptides, but not of HNE-modified peptides, were reduced significantly and dose-dependently. Concomitantly, the formation of PM-ONE adducts increased in a dose-dependent manner. The inhibition effect of PM was also confirmed in the cell system subjected to oxidative stress. Our results demonstrate that PM can inhibit lipid hydroperoxide-derived damage to proteins by trapping ONE preferentially, and the resulting PM-ONE adducts can be used as a dosimeter for ONE production to determine the levels of lipid peroxidation.

The current study originates from the assumption that, in tumors, levels of naturally occurring pyridoxal 5'-phosphate (PLP) are too small to allow conversion of tetra hydro pteroylglutamate (H4PteGlu) into methylene tetra hydro pteroylglutamate (CH2-H4PteGlu) in amounts required to improve inhibition of thymidylate synthase by 5-fluorouracil (FUra) through ternary complex stabilization. The hypothesis relates to the low affinity for cofactor of the PLP-dependent serine hydroxymethyl transferase (SHMT), the enzyme that catalyzes formation of CH2-H4PteGlu by transfer of the Cβ of serine to H4PteGlu. Intracellular concentrations of PLP are smaller than the dissociation constant of SHMT for cofactor, which suggests that enzyme activity should be sensitive to PLP level changes. Three cancer cell lines were supplemented with PLP to investigate the influence of this cofactor on FUra cytotoxicity. Cells were exposed to FUra, FUra and folinic acid (FA), FUra and PLP, and FUra combined with both FA and PLP. The median-effect principle for concentration-effect analysis and combination indices were used to determine interactions on cytotoxicity. FUra cytotoxicity in vitro was enhanced by FA and PLP in tandem. Synergistic cytotoxic interaction of FUra with FA and PLP was demonstrated in HT29 and L1210 cells. Summation was found in HCT116 cells. Parenteral pyridoxamine was administered in mice to explore erythrocyte production of PLP in vivo. Cofactor attained levels in the range of the KD for binding to SHMT, and it was rapidly cleared from cells. Pharmacokinetics of pyridoxamine suggests that modulation of FUra by vitamin B6 could be achieved in vivo.

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of Mycobacterium tuberculosis (Mtb). PLP functions as a cofactor for 58 putative PLP-binding proteins encoded by the Mtb genome and could also act as a potential antioxidant. De novo biosynthesis of PLP in Mtb takes place through the 'deoxyxylulose 5'-phosphate (DXP)-independent' pathway, whereas PdxH enzymes, possessing pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPOx) activity, are involved in the PLP salvage pathway. In this study, we demonstrate that the annotated PdxH enzymes from various mycobacterial species are bona fide members of the classical PNPOx enzyme family, capable of producing PLP using both pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) substrates.

Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5'-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5'-phosphate (PMP), pyridoxine 5'-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism.

For the aromatic OH group in pyridoxine, pyridoxal, pyridoxamine (vitamin B6 components) and their metabolite pyridoxic acid, reaction enthalpies related to various mechanisms of primary antioxidant action, i.e. Hydrogen Atom Transfer (HAT), Single Electron Transfer–Proton Transfer (SET–PT) and Sequential Proton-Loss Electron-Transfer (SPLET) were investigated. B3LYP, M05-2X and M06-2X functionals were employed in this study. Two solvation models were used for the solution-phase calculations in benzene and water: integral equation formalism polarized continuum model, IEF–PCM, as well as newer SMD method. All computational approaches provide identical trends. Comparison of found results with data obtained previously for phenol, α-tocopherol and flavonols indicates that vitamin B6 may show weaker antioxidant effect than naturally occurring phenolic antioxidants.

BACKGROUND Little is known about how vitamins can affect the peroxidation and stability of polyunsaturated fatty acids in cooked foods. Thus the effects of 15 vitamins on toxic malondialdehyde (MDA) formation in cooked beef patties were examined with the application of solid phase extraction and thiobarbituric acid (TBA) analysis by HPLC-DAD. The polyunsaturated fatty acid profiles in cooked beef patties treated with some vitamins were further compared with that of control sample (no vitamin addition) by GC-MS analysis. RESULTS Pyridoxamine, pyridoxine, retinoic acid, α-tocopherol and L-ascorbic acid exhibited significant effects lowering the amount of MDA. It was further discovered that retinoic acid, α-tocopherol and l-ascorbic acid could help preserve polyunsaturated fatty acids, while pyridoxamine addition actually showed no effect upon the retention of most of the tested polyunsaturated fatty acids, even lowering the content of arachidonic acid. Further LC-MS analysis demonstrated that pyridoxamine could directly react with MDA via an addition reaction. The reaction involves a nucleophilic attack of pyridoxamine's free amine group on one of the aldehyde functional groups of MDA to form a new adduct, and may accelerate lipid peroxidation with the loss of more polyunsaturated fatty acids. CONCLUSION Some vitamins may directly participate in lipid peroxidation and affect food quality. © 2015 Society of Chemical Industry

Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes 64: 2242–2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy.