B6 Vitamers Research Articles

Purine limitation prevents the exogenous pyridoxal 5'-phosphate accumulation of Salmonella enterica yggS mutants.

Pyridoxal 5'-phosphate is the active form of vitamin B6 and is an essential cofactor in all domains of life. PLP can be synthesized de novo or salvaged from the environment from one of the six B6 vitamers. B6 vitamer levels appear to be tightly regulated, and alterations in their levels can have deleterious effects, most notably being the development of B6-dependent epilepsy in humans. YggS homologs are broadly conserved across multiple organisms and considered to be involved in maintaining B6 homeostasis, though no specific mechanism has been defined. The current study showed that the exogenous accumulation of PLP caused by a lack of YggS can be prevented by purine limitation. The demonstration that purine limitation impacts exogenous PLP accumulation separates one consequence of a yggS mutation for further study and contributes to continuing efforts to define the biochemical and physiological roles of the COG0325 family of proteins.

Exploration of Vitamin B6-Based Redox-Active Pyridinium Salts Towards the Application in Aqueous Organic Flow Batteries.

Pyridoxal hydrochloride, a vitamin B6 vitamer, was synthetically converted to a series of diverse redox-active benzoyl pyridinium salts. Cyclic voltammetry studies demonstrated redox reversibility under basic conditions, and two of the most promising salts were subjected to laboratory-scale flow battery tests involving galvanostatic cycling at 10 mM in 0.1 M NaOH. In these tests, the battery was charged completely, corresponding to the transfer of two electrons to the electrolyte, but no discharge was observed. Both CV analysis and electrochemical simulations confirmed that the redox wave observed in the experimental voltammograms corresponds to a two-electron process. To explain the irreversibility in the battery tests, we conducted bulk electrolysis with the benzoyl pyridinium salts, affording the corresponding benzylic secondary alcohols. Computational studies suggest that the reduction proceeds in three consecutive steps: first electron transfer (ET), then proton-coupled electron transfer (PCET) and finally proton transfer (PT) to give the secondary alcohol. 1H NMR deuterium exchange studies indicated that the last PT step is not reversible in 0.1 M NaOH, rendering the entire redox process irreversible. The apparent reversibility observed in CV at the basic media likely arises from the slow rate of the PT step at the timescale of the measurement.

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.

Vitamin B6 is governed by the local compartmentalization of metabolic enzymes during growth.

Vitamin B6 is a vital micronutrient across cell types and tissues, and dysregulated B6 levels contribute to human disease. Despite its importance, how B6 vitamer levels are regulated is not well understood. Here, we provide evidence that B6 dynamics are rapidly tuned by precise compartmentation of pyridoxal kinase (PDXK), the rate-limiting B6 enzyme. We show that canonical Wnt rapidly led to the accumulation of inactive B6 by shunting cytosolic PDXK into lysosomes. PDXK was modified with methyl-arginine Degron (MrDegron), a protein tag for lysosomes, which enabled delivery via microautophagy. Hyperactive lysosomes resulted in the continuous degradation of PDXK and B6 deficiency that promoted proliferation in Wnt-driven colorectal cancer (CRC) cells. Pharmacological or genetic disruption of the coordinated MrDegron proteolytic pathway was sufficient to reduce CRC survival in cells and organoid models. In sum, this work contributes to the repertoire of micronutrient-regulated processes that enable cancer cell growth and provides insight into the functional impact of B6 deficiencies for survival.

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 HEK293cells 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 HEK293cells 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 HEK293cells. 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.

N-Alkylated Pyridoxal Derivatives as Negative Electrolyte Materials for Aqueous Organic Flow Batteries: Computational Screening.

N-functionalized pyridinium frameworks derived from the three major vitamers of vitamin B6, pyridoxal, pyridoxamine and pyridoxine, have been screened computationally for consideration as negative electrode materials in aqueous organic flow batteries. A molecular database including the structure and the one-electron standard reduction potential of related pyridinium derivatives has been generated using a computational protocol that combines semiempirical and DFT quantum chemical methods. The predicted reduction potentials span a broad range for the investigated pyridinium frameworks, but pyridoxal derivatives, particularly those involving electron withdrawing substituents, have potentials compatible with the electrochemical stability window of aqueous electrolytes. The stability of radicals formed upon one-electron reduction has been analyzed by a new computational tool proposed recently for large-scale computational screening.

Clinical and biochemical impact of vitamin B6 deficiency in primary sclerosing cholangitis before and after liver transplantation

We previously demonstrated that people with primary sclerosing cholangitis (PSC) had reduced gut microbial capacity to produce active vitamin B6 (pyridoxal 5'-phosphate [PLP]), which corresponded to lower circulating PLP levels and poor outcomes. Here, we define the extent and biochemical and clinical impact of vitamin B6 deficiency in people with PSC from several centers before and after liver transplantation (LT). We used targeted liquid chromatography-tandem mass spectrometry to measure B6 vitamers and B6-related metabolic changes in blood from geographically distinct cross-sectional cohorts totaling 373 people with PSC and 100 healthy controls to expand on our earlier findings. Furthermore, we included a longitudinal PSC cohort (n= 158) sampled prior to and serially afterLT, and cohorts of people with inflammatory bowel disease (IBD) without PSC (n= 51) or with primary biliary cholangitis (PBC) (n= 100), as disease controls. We used Cox regression to measure the added value of PLP to predict outcomes before and after LT. In different cohorts, 17-38% of people with PSC had PLP levels below the biochemical definition of a vitamin B6 deficiency. The deficiency was more pronounced in PSC than in IBD without PSC and PBC. Reduced PLP was associated with dysregulation of PLP-dependent pathways. The low B6 status largely persisted after LT. Low PLP independently predicted reduced LT-free survival in both non-transplanted people with PSC and in transplant recipients with recurrent disease. Low vitamin B6 status with associated metabolic dysregulation is a persistent feature of PSC. PLP was a strong prognostic biomarker for LT-free survival both in PSC and recurrent disease. Our findings suggest that vitamin B6 deficiency modifies the disease and provides a rationale for assessing B6 status and testing supplementation. We previously found that people with PSC had reduced gut microbial potential to produce essential nutrients. Across several cohorts, we find that the majority of people with PSC are either vitamin B6 deficient or have a marginal deficiency, which remains prevalent even after liver transplantation. Low vitamin B6 levels strongly associate with reduced liver transplantation-free survival as well as deficits in biochemical pathways dependent on vitamin B6, suggesting that the deficiency has a clinical impact on the disease. The results provide a rationale for measuring vitamin B6 and to investigate whether vitamin B6 supplementation or modification of the gut microbial community can help improve outcomes for people with PSC.

Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942.

The cyanobacterial protein PipY belongs to the Pyridoxal-phosphate (PLP)-binding proteins (PLPBP/COG0325) family of pyridoxal-phosphate-binding proteins, which are represented in all three domains of life. These proteins share a high degree of sequence conservation, appear to have purely regulatory functions, and are involved in the homeostasis of vitamin B6 vitamers and amino/keto acids. Intriguingly, the genomic context of the pipY gene in cyanobacteria connects PipY with PipX, a protein involved in signaling the intracellular energy status and carbon-to-nitrogen balance. PipX regulates its cellular targets via protein-protein interactions. These targets include the PII signaling protein, the ribosome assembly GTPase EngA, and the transcriptional regulators NtcA and PlmA. PipX is thus involved in the transmission of multiple signals that are relevant for metabolic homeostasis and stress responses in cyanobacteria, but the exact function of PipY is still elusive. Preliminary data indicated that PipY might also be involved in signaling pathways related to the stringent stress response, a pathway that can be induced in the unicellular cyanobacterium Synechococcus elongatus PCC7942 by overexpression of the (p)ppGpp synthase, RelQ. To get insights into the cellular functions of PipY, we performed a comparative study of PipX, PipY, or RelQ overexpression in S. elongatus PCC7942. Overexpression of PipY or RelQ caused similar phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules. In contrast, PipX overexpression decreased cell length, indicating that PipX and PipY play antagonistic roles on cell elongation or cell division. Since ppGpp levels were not induced by overexpression of PipY or PipX, it is apparent that the production of polyphosphate in cyanobacteria does not require induction of the stringent response.

Pyridoxamine reduces methylglyoxal and markers of glycation and endothelial dysfunction, but does not improve insulin sensitivity or vascular function in abdominally obese individuals: A randomized double-blind placebo-controlled trial.

To investigate the effects of pyridoxamine (PM), a B6 vitamer and dicarbonyl scavenger, on glycation and a large panel of metabolic and vascular measurements in a randomized double-blindplacebo-controlled trial in abdominally obese individuals. Individuals (54% female; mean age 50 years; mean body mass index 32 kg/m2 ) were randomized to an 8-week intervention with either placebo (n=36), 25 mg PM (n=36) or 200 mg PM (n=36). We assessed insulin sensitivity, β-cell function, insulin-mediated microvascular recruitment, skin microvascular function, flow-mediated dilation, and plasma inflammation and endothelial function markers. PM metabolites, dicarbonyls and advanced glycation endproducts (AGEs) were measured using ultra-performance liquid chromatography tandem mass spectrometry. Treatment effects were evaluated by one-way ANCOVA. In the high PM dose group, we found a reduction of plasma methylglyoxal (MGO) and protein-bound Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine(MG-H1), as compared to placebo. We found a reduction of the endothelial dysfunction marker soluble vascular celladhesion molecule-1 (sVCAM-1) in the low and high PM dose group and of soluble intercellularadhesion molecule-1(sICAM-1) in the high PM dose, as compared to placebo. We found no treatment effects on insulin sensitivity, vascular function or other functional outcome measurements. This study shows that PM is metabolically active and reduces MGO, AGEs, sVCAM-1 and sICAM-1, but does not affect insulin sensitivity and vascular function in abdominally obese individuals. The reduction in adhesion markers is promising because these are important in the pathogenesis of endothelial damage and atherosclerosis.

Pyridoxal 5'-phosphate synthesis and salvage in Bacteria and Archaea: predicting pathway variant distributions and holes.

Pyridoxal 5’-phosphate or PLP is a cofactor derived from B6 vitamers and essential for growth in all known organisms. PLP synthesis and salvage pathways are well characterized in a few model species even though key components, such as the vitamin B6 transporters, are still to be identified in many organisms including the model bacteria Escherichia coli or Bacillus subtilis . Using a comparative genomic approach, PLP synthesis and salvage pathways were predicted in 5840 bacterial and archaeal species with complete genomes. The distribution of the two known de novo biosynthesis pathways and previously identified cases of non-orthologous displacements were surveyed in the process. This analysis revealed that several PLP de novo pathway genes remain to be identified in many organisms, either because sequence similarity alone cannot be used to discriminate among several homologous candidates or due to non-orthologous displacements. Candidates for some of these pathway holes were identified using published TnSeq data, but many remain. We find that ~10 % of the analysed organisms rely on salvage but further analyses will be required to identify potential transporters. This work is a starting point to model the exchanges of B6 vitamers in communities, predict the sensitivity of a given organism to drugs targeting PLP synthesis enzymes, and identify numerous gaps in knowledge that will need to be tackled in the years to come.

국가식품성분표 개정을 위한 국내 다빈도 섭취 계육 및 계육 조리가공품의 B6 및 B12 vitamers (pyridoxine, pyridoxal, pyridoxamine, cobalamins) 분석

In this study, vitamers of vitamins B6 (pyridoxine; PN, pyridoxal; PL, pyridoxamine; PM) and B12 (cobalamins) in cooked or processed chicken (n=21) were analyzed and the analytical performance parameters were evaluated. The levels of B6 and B12 vitamers were significantly different in terms of the breeds, cooking method, and the parts of the chicken (p⟨0.05). Ogolgye (boiled) (61.48 μg/100 g) and roasted chicken wings (131.94 μg/100 g) showed the highest levels of total vitamin B6 (PN+PL+PM) among the four breeds of chiken and the cooked or processed chiken, respectively. For cyanocobalamin, Korean native chicken (0.40 μg/100 g) and chicken skewers (0.68 μg/100 g) showed the highest levels among the four breeds of chicken and the cooked or processed chiken, respectively. Analysis of B6 vitamers using high performance liquid chromatography-florescence detector (HPLC-FLD) showed a coefficient of variation (CV) of 0.4-4.6% for repeatability and 4.2-5.9% for reproducibility, showing good precision. Likewise, vitamin B12 analysis using immunoaffinity-HPLC-photodiode array detector (PDA) showed a CV of 5.7% for repeatability and 5.9% for reproducibility. Recoveries of B6 and B12 vitamers were 94.3-100.2%, showing good accuracy. Unlike many previous studies that evaluated PN only, this study provides a more accurate estimation of the total B6 content of cooked or processed chiken, including the contents of PN, PL, and PM, which can be used to revise the Korean food composition table.

Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability.

The pyridoxal 5′‐phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP‐dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP‐dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

The Conserved Family of the Pyridoxal Phosphate-Binding Protein (PLPBP) and Its Cyanobacterial Paradigm PipY.

The PLPBP family of pyridoxal phosphate-binding proteins has a high degree of sequence conservation and is represented in all three domains of life. PLPBP members, of which a few representatives have been studied in different contexts, are single-domain proteins with no known enzymatic activity that exhibit the fold type III of PLP-holoenzymes, consisting in an α/β barrel (TIM-barrel), where the PLP cofactor is solvent-exposed. Despite the constant presence of cofactor PLP (a key catalytic element in PLP enzymes), PLPBP family members appear to have purely regulatory functions affecting the homeostasis of vitamin B6 vitamers and amino/keto acids. Perturbation of these metabolites and pleiotropic phenotypes have been reported in bacteria and zebrafish after PLPBP gene inactivation as well as in patients with vitamin B6-dependent epilepsy that results from loss-of-function mutations at the PLPBP. Here, we review information gathered from diverse studies and biological systems, emphasizing the structural and functional conservation of the PLPBP members and discussing the informative nature of model systems and experimental approaches. In this context, the relatively high level of structural and functional characterization of PipY from Synechococcus elongatus PCC 7942 provides a unique opportunity to investigate the PLPBP roles in the context of a signaling pathway conserved in cyanobacteria.

Validated UPLC-MS/MS method for the analysis of vitamin B6 pyridoxal 5́-phosphate, pyridoxal, pyridoxine, pyridoxamine, and pyridoxic acid in human cerebrospinal fluid

Vitamin B6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories.Until today, vitamin B6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory.

Investigation of the vitamins B1, B2, and B6 vitamers bioaccessibilities of canned, dried legumes after in vitro gastrointestinal digestion system

Legumes are rich in minerals, B group vitamins, fiber, and protein. Intake of essential nutrients is vital in adequate and balanced nutrition. As it is crucial to evaluate final nutrient amounts, cooking losses and bioaccessibility values are determinant factors. This study investigates the quantity and vitamins B1, B2, and B6 vitamers bioaccessibilities in different dried, canned legume samples using an in vitro digestion model. High-performance liquid chromatography was used to determine the amount of each vitamin. Vitamin B1 bioaccessibility in canned legumes was found above 72% except for red lentils (23%), vitamin B2 bioaccessibility was above 63% except for green lentils (44%), while total vitamin B6 bioaccessibility (57%) was lower than vitamins B1 and B2. The form of pyridoxine with the highest bioaccessibility for vitamin B6 forms was found between 66 and 89%, except for peas and red lentils. Besides, pyridoxamine form bioaccessibility was very low compared to pyridoxal form. We believe bioaccessibility might relate to temperature, pH, bonds with polypeptides and polysaccharides, and dietary fibers. As seen, the concept of bioaccessibility gains importance in the final nutrient amount.

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.

The Nitrogen Atom of Vitamin B6 Is Essential for the Catalysis of Radical Aminomutases.

Radical aminomutases are pyridoxal 5′-phosphate (PLP, a B6 vitamer)-dependent enzymes that require the generation of a 5′-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases has not been investigated extensively yet. We report an alternative synthetic procedure to provide easy access to 1-deazaPLP (dAPLP), an isosteric analog of PLP which acts as a probe for studying the role of the nitrogen atom. Our results revealed that lysine 5,6-aminomutase (5,6-LAM), a radical aminomutase, reconstituted with dAPLP cannot turn over a substrate, demonstrating that the nitrogen atom is essential for radical aminomutases. In contrast, biochemical and spectroscopic studies on the S238A variant reconstituted with PLP revealed a minuscule loss of activity. This apparent anomaly can be explained by a water-mediated rescue of activity in S238A, as if mimicking the active site of lysine 2,3-aminomutase. This study leads to a better comprehension of how enzymes harness the optimum capability of PLP to realize catalysis.

Increased vitamin B6 turnover is associated with greater mortality risk in the general US population: A prospective biomarker study

Vitamin B6 status and mortality risk are inversely associated in different patient groups, while prospective studies in the general population are lacking. Here, for the first time, we evaluated the association between biomarkers of vitamin B6 status and mortality risk in a large population-based study. The vitamin B6 vitamers pyridoxal-5'-phosphat (PLP) and 4-pyridoxic acid (4-PA) were measured by high-performance liquid chromatography in the National Health and Nutrition Examination Survey (NHANES) between 2005 and 2010. Participants' vital status and causes of death were recorded until December 2015. Multivariable Cox regression analyses were carried out to estimate Hazard Ratios (HRs) and 95% confidence intervals (CIs) of mortality across quintiles of PLP, 4-PA, and the ratio of 4-PA and PLP. Out of 15,304 study participants aged between 20 and 85 years at baseline, 1666 (7.7%) died during a median follow-up time of 7.8 years. An inverse association between PLP and mortality was found in a multivariable model adjusted for socioeconomic and lifestyle factors but became statistically non-significant upon adjustment for routine biomarkers (C-reactive protein, creatinine, albumin, and alkaline phosphatase). There was a significant linear trend for a positive association between 4-PA levels and mortality risk in the fully adjusted regression model, although a comparison of extreme quintiles (quintile 5 vs. quintile 1) did not show a significant difference (HRQ5vs.Q1 (95% CI): 1.19 (0.93, 1.51), plinear trend=0.02). A positive association between the 4-PA/PLP ratio and all-cause mortality was observed in the multivariable model, with an HRsQ5vs.Q1 of 1.45 (95% CI: 1.14, 1.85; plinear trend<0.0001). There were no significant associations between the biomarkers and cardiovascular or cancer mortality. The association between 4-PA/PLP and mortality risk was heterogeneous across age groups, and only statistically significant among participants older than 65 years at baseline (HRQ5vs.Q1 (95% CI): 1.72 (1.29, 2.29), plinear trend<0.0001). In this group, 4-PA/PLP was also associated with cancer mortality, with an HR Q5vs.Q1 of 2.16 (1.20, 3.90), plinear trend=0.02). Increased vitamin B6 turnover, as indicated by a higher 4-PA/PLP ratio, was associated with all-cause and cancer mortality among the older U.S. general population. Intervention trials are needed to assess whether older individuals with a high 4-PA/PLP ratio would benefit from increased vitamin B6 intake.

Loss of a pyridoxal-phosphate phosphatase rescues Arabidopsis lacking an endoplasmic reticulum ATP carrier.

The endoplasmic reticulum (ER)-located ATP/ADP-antiporter (ER-ANT1) occurs specifically in vascular plants. Structurally different transporters mediate energy provision to the ER, but the cellular function of ER-ANT1 is still unknown. Arabidopsis (Arabidopsis thaliana) mutants lacking ER-ANT1 (er-ant1 plants) exhibit a photorespiratory phenotype accompanied by high glycine levels and stunted growth, pointing to an inhibition of glycine decarboxylase (GDC). To reveal whether it is possible to suppress this marked phenotype, we exploited the power of a forward genetic screen. Absence of a so far uncharacterized member of the HaloAcid Dehalogenase (HAD)-like hydrolase family strongly suppressed the dwarf phenotype of er-ant1 plants. Localization studies suggested that the corresponding protein locates to chloroplasts, and activity assays showed that the enzyme dephosphorylates, with high substrate affinity, the B6 vitamer pyridoxal 5'-phosphate (PLP). Additional physiological experiments identified imbalances in vitamin B6 homeostasis in er-ant1 mutants. Our data suggest that impaired chloroplast metabolism, but not decreased GDC activity, causes the er-ant1 mutant dwarf phenotype. We present a hypothesis, setting transport of PLP by ER-ANT1 and chloroplastic PLP dephosphorylation in the cellular context. With the identification of this HAD-type PLP phosphatase, we also provide insight into B6 vitamer homeostasis.

PDX1.1-dependent biosynthesis of vitamin B6 protects roots from ammonium-induced oxidative stress

Despite serving as a major inorganic nitrogen source for plants, ammonium causes toxicity at elevated concentrations, inhibiting root elongation early on. While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species (ROS) in roots, it remains unclear about the mechanisms underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium. In this study, we demonstrate that ammonium-induced apoplastic acidification co-localizes with Fe precipitation and hydrogen peroxide (H2O2) accumulation along the stele of the elongation and differentiation zone in root tips, indicating Fe-dependent ROS formation. By screening ammonium sensitivity in T-DNA insertion lines of ammonium-responsive genes, we identified PDX1.1, which is upregulated by ammonium in the root stele and whose product catalyzes de novo biosynthesis of vitamin B6. Root growth of pdx1.1 mutants is hypersensitive to ammonium, while chemical complementation or overexpression of PDX1.1 restores root elongation. This salvage strategy requires non-phosphorylated forms of vitamin B6 that are able to quench ROS and rescue root growth from ammonium inhibition. Collectively, these results suggest that PDX1.1-mediated synthesis of non-phosphorylated B6 vitamers acts as a primary strategy to protect roots from ammonium-dependent ROS formation.