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Objectives: Because angiotensin (Ang) II is an essential vasoconstrictive peptide, we analyzed the impact of its post‐translational modification to pyruvamide–Ang II (Ang P) by pyridoxal‐5′‐phosphate (PLP) on blood pressure. PLP is a less expensive vitamin B6 derivative and, therefore, could be a cost‐effective drug against hypertension. Methods: Effect of Ang P on calcium ion entry into vascular smooth muscle cells (VSMCs) was analyzed. Binding affinity of Ang P to angiotensin II type 1 receptor (AT1R) was measured. Vasoconstrictive effect of Ang P was investigated using the bioassay of isolated perfused rat kidneys. Spontaneously hypertensive rats (SHR) were administered PLP. Additionally, Wistar Kyoto rats (WKY) received Ang II and PLP. Blood pressure was measured time‐dependently. Results: Ang II, incubated with PLP, was post‐translationally modified to Ang P. Calcium ion entry in VSMCs was significantly lower with Ang P compared to Ang II. Binding affinity of Ang P to AT1R was lower compared to Ang II. Perfusion pressure of isolated perfused rat kidneys increased less by Ang P than by Ang II. Blood pressure of SHR treated with PLP decreased significantly. Blood pressure of WKY rats treated with Ang II was increased to hypertensive values, whereas blood pressure of WKY rats cotreated with Ang II and PLP was not. Conclusion: PLP induces a post‐translational modification of Ang II decreasing blood pressure in rats. Assuming that increased PLP intake in the form of vitamin B6 might reduce blood pressure in hypertensive patients, PLP might be a cost‐effective drug against hypertension. [ABSTRACT FROM AUTHOR]

LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the peptidase CLPP also act in the matrix, especially during stress periods, but their substrates are poorly defined. Mammalian CLPP deletion triggers infertility, deafness, growth retardation, and cGAS-STING-activated cytosolic innate immunity. CLPX mutations impair heme biosynthesis and heavy metal homeostasis. CLPP and CLPX are conserved from bacteria to humans, despite their secondary role in proteolysis. Based on recent proteomic–metabolomic evidence from knockout mice and patient cells, we propose that CLPP acts on phase-separated ribonucleoprotein granules and CLPX on multi-enzyme condensates as first-aid systems near the inner mitochondrial membrane. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, mitochondrial RNA granules and nucleoids, and the D-foci-mediated degradation of toxic double-stranded mtRNA/mtDNA. Unfolding multi-enzyme condensates, CLPX maximizes PLP-dependent delta-transamination and rescues malformed nascent peptides. Overall, their actions occur in granules with multivalent or hydrophobic interactions, separated from the aqueous phase. Thus, the role of CLPXP in the matrix is compartment-selective, as other mitochondrial peptidases: MPPs at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space. [ABSTRACT FROM AUTHOR]

Exploring the link between genetic polymorphisms in folate metabolism genes (MTHFR, MTR, and MTRR) and cardiovascular disease (CVD), this study evaluates the effect of B vitamin supplements (methylfolate, pyridoxal-5′-phosphate, and methylcobalamin) on homocysteine and lipid levels, potentially guiding personalized CVD risk management. In a randomized, double-blind, placebo-controlled trial, 54 patients aged 40–75 with elevated homocysteine and moderate LDL-C levels were divided based on MTHFR, MTR, and MTRR genetic polymorphisms. Over six months, they received either a combination of methylfolate, P5P, and methylcobalamin, or a placebo. At the 6 months follow-up, the treatment group demonstrated a significant reduction in homocysteine levels by 30.0% (95% CI: −39.7% to −20.3%) and LDL-C by 7.5% (95% CI: −10.3% to −4.7%), compared to the placebo (p < 0.01 for all). In the subgroup analysis, Homozygous Minor Allele Carriers showed a more significant reduction in homocysteine levels (48.3%, 95% CI: −62.3% to −34.3%, p < 0.01) compared to mixed allele carriers (18.6%, 95% CI: −25.6% to −11.6%, p < 0.01), with a notable intergroup difference (29.7%, 95% CI: −50.7% to −8.7%, p < 0.01). LDL-C levels decreased by 11.8% in homozygous carriers (95% CI: −15.8% to −7.8%, p < 0.01) and 4.8% in mixed allele carriers (95% CI: −6.8% to −2.8%, p < 0.01), with a significant between-group difference (7.0%, 95% CI: −13.0% to −1.0%, p < 0.01). Methylfolate, P5P, and methylcobalamin supplementation tailored to genetic profiles effectively reduced homocysteine and LDL-C levels in patients with specific MTHFR, MTR, and MTRR polymorphisms, particularly with homozygous minor allele polymorphisms. [ABSTRACT FROM AUTHOR]

Background: Inflammation can increase vitamin B6 uptake and catabolism. Higher vitamin B6 turnover [4-pyridoxic acid (4-PA)/pyridoxal 5'-phosphate (PLP) ratio], was associated with mortality risk in the general population. Objectives: We aimed to investigate the association between 4-PA/PLP and long-term mortality in patients with type 2 diabetes mellitus (T2DM), an inflammatory disease. Methods: In this prospective cohort study from the National Health and Nutrition Examination Survey (NHANES) cycles 2005-2010, the concentrations of 4-PA and PLP in plasma were measured using high-performance liquid chromatography, with mortality data updated to 31 December 2019. We included 2074 patients with T2DM aged between 20 and 85 y at baseline. Results: There were 739 deaths among 2279 patients with T2DM with a median follow-up of 11.83 y. In the age- and sex-adjusted COX model (model 1), 4-PA/PLP was positively associated with mortality in patients with T2DM [hazard ratio (HR) and 95% confidence interval (CI) highest compared with lowest quartiles: 35.55 (18.29, 69.09); P < 0.001], and in model 3, which was adjusted for demographics as well as inflammation, nutrition, and renal function, high 4-PA/PLP concentrations remained an independent risk factor for mortality in patients with T2DM [HR (95% CI) highest compared with lowest quartiles: 5.03 (2.46, 10.30); P < 0.001]. In restricted cubic spline (RCS), the link between 4-PA/PLP and all-cause mortality displays a positive correlation. Patients with died within the previous 2 y were excluded, the sensitivity analysis had no effect on the association between 4-PA/PLP and mortality in patients with T2DM. Finally, comparable results were found in subgroup analyses of specific-cause mortality. Conclusion: Higher vitamin B6 turnover is associated with long-term mortality risk in patients with T2DM. 4-PA/PLP may serve as a convenient prognostic marker in T2DM management. [ABSTRACT FROM AUTHOR]

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Simple Summary: The molecular mechanisms of Charcot–Marie–Tooth (CMT) disease, involving impaired vitamin metabolism and/or actions, are considered in light of the potential therapeutic actions of vitamins B1, B3 and B6 in the disease. Although the different disease cues merge into similar symptoms, identification of disease-specific molecular mechanisms is necessary to develop tailored treatments in personalized medicine. Understanding the molecular mechanisms of neurological disorders is necessary for the development of personalized medicine. When the diagnosis considers not only the disease symptoms, but also their molecular basis, treatments tailored to individual patients may be suggested. Vitamin-responsive neurological disorders are induced by deficiencies in vitamin-dependent processes. These deficiencies may occur due to genetic impairments of proteins whose functions are involved with the vitamins. This review considers the enzymes encoded by the DHTKD1, PDK3 and PDXK genes, whose mutations are observed in patients with Charcot–Marie–Tooth (CMT) disease. The enzymes bind or produce the coenzyme forms of vitamins B1 (thiamine diphosphate, ThDP) and B6 (pyridoxal-5′-phosphate, PLP). Alleviation of such disorders through administration of the lacking vitamin or its derivative calls for a better introduction of mechanistic knowledge to medical diagnostics and therapies. Recent data on lower levels of the vitamin B3 derivative, NAD+, in the blood of patients with CMT disease vs. control subjects are also considered in view of the NAD-dependent mechanisms of pathological axonal degeneration, suggesting the therapeutic potential of vitamin B3 in these patients. Thus, improved diagnostics of the underlying causes of CMT disease may allow patients with vitamin-responsive disease forms to benefit from the administration of the vitamins B1, B3, B6, their natural derivatives, or their pharmacological forms. [ABSTRACT FROM AUTHOR]

This study was to assess the association between vitamin B6 turnover rate and mortality in hypertensive adults. Vitamin B6 status including serum pyridoxal-5′-phosphate (PLP) levels, serum 4-pyridoxal acid (4-PA) levels, and vitamin B6 turnover rate (4-PA/PLP) were obtained from the 2005–2010 National Health and Nutrition Examination Survey (NHANES) dataset of hypertensive adults with follow-up through December 30, 2019. Using Cox proportional risk regression models, Hazard ratios (HRs) and 95% confidence intervals (CIs) were analyzed for PLP, 4-PA and 4-PA/PLP quartiles in relation to cardiovascular and all-cause mortality. A total of 5434 participants were included in this study (mean age, 58.48 years; 50.4% men), and the median 4-PA/PLP was 0.75. The median follow-up time was 11.0 years, with 375 and 1387 cardiovascular and all-cause deaths, respectively. In multivariate COX regression models, PLP was negatively associated with cardiovascular mortality (HR [95% CI] quartile 4 vs. 1: 0.66 [0.47–0.94], P trend = 0.03) and 4-PA/PLP was positively associated with cardiovascular mortality (HR [95% CI] quartile 4 vs.1: 1.80 [1.21–2.67], P trend = 0.01). Similarly, the higher the quartile of PLP, the lower the risk of all-cause mortality (HR [95% CI] quartile 4 vs. 1: 0.67 [0.56–0.80], P trend < 0.01). The higher the quartile of 4-PA and 4-PA/PLP, the higher the risk of all-cause mortality (HR [95% CI] quartile 4 vs. 1: 1.22 [1.01–1.48], P trend < 0.01; and 2.09 [1.71–2.55], P trend < 0.01). The findings suggested that higher vitamin B6 turnover rate was associated with an increased risk of cardiovascular and all-cause mortality in hypertensive adults. • Vitamin B6 status was associated with cardiovascular and all-cause mortality in a hypertensive population. • Higher vitamin B6 turnover rate was associated with an increased risk of mortality in hypertensive adults. • Vitamin B6 turnover may be a biomarker of disease and mortality risk. [ABSTRACT FROM AUTHOR]

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5′-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination. [ABSTRACT FROM AUTHOR]

Cystathionine γ‐lyase (CGL) is a PLP‐dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL‐catalyzed process consists of an α,γ‐elimination reaction that breaks down cystathionine into cysteine, α‐ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2S). Importantly, inhibition of the enzyme and consequently of its H2S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as Toxoplasma gondii, the causative agent of toxoplasmosis, encode a CGL enzyme (TgCGL) that almost exclusively catalyzes the canonical process, with only minor reactivity to cysteine. Interestingly, the substitution of N360 by a serine (the equivalent amino acid residue in the human enzyme) at the active site changes the specificity of TgCGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the CγS and the CβS bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme‐substrate specificity, we have elucidated the crystal structures of native TgCGL and the variant TgCGL‐N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor d,l‐propargylglycine (PPG). Our structures reveal the binding mode of each molecule within the catalytic cavity and help explain the inhibitory behavior of cysteine and PPG. A specific inhibitory mechanism of TgCGL by PPG is proposed. PDB Code(s): 7NL1;8BIS;8BIU;8BIV;8BIW;8BIX;8BIZ [ABSTRACT FROM AUTHOR]

Transaminases are pyridoxal 5'‐phosphate (PLP)‐dependent enzymes that transfer amino‐functions. The transaminase from Silicibacter pomeroyi (SpATA) exhibits a broad substrate spectrum. In this work we examined the effect of different conditions (light, buffer and PLP‐concentration) on the stability of SpATA, as well as the causes for these effects. The enzyme was stored either in TRIS or CHES with 0–10 mM added PLP at 22 °C. The samples were either kept dark or they were exposed to light. The results show that invariably, all samples kept in darkness exhibited longer half‐life times than the ones exposed to light. An increase in the half‐life from 8 h to 720 h could be achieved solely by keeping the sample dark. Especially samples in CHES buffer inactivated faster in light the more PLP was present, due to the degradation of PLP. In TRIS however, an imine‐bond between TRIS and PLP protects PLP from degradation. [ABSTRACT FROM AUTHOR]

d‐Glucosaminate‐6‐phosphate ammonia‐lyase (DGL) catalyzes the conversion of d‐glucosaminate‐6‐phosphate to 2‐keto‐3‐deoxyglutarate‐6‐phosphate, with stereospecific protonation of C‐3 of the product. The crystal structure of DGL showed that His‐163 could serve as the proton donor. H163A mutant DGL is fully active in the steady‐state reaction, and the pre‐steady‐state kinetics are very similar to those of wild‐type DGL. However, H163A DGL accumulates a transient intermediate with λmax at 293 nm during the reaction that is not seen with wild‐type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D2O shows that the product is a mixture of deuterated diastereomers at C‐3. These results establish that His‐163 is the proton donor in the reaction mechanism of DGL. [ABSTRACT FROM AUTHOR]

A rare subset of vitamin B6 responsive seizure disorders does not respond to pyridoxine, and requires the active form of vitamin B6, pyridoxal-5′-phosphate (PLP), to maintain seizure control. Patients with PLP-responsive seizures are dependent on chronic PLP treatment, yet no licensed PLP product is available. PLP food supplements, a product category regulated less stringently than medication, may prove of insufficient effectiveness and safety. Here we describe and discuss three patient scenarios which illustrate this conundrum. Medical and laboratory records were reviewed with retrospective extraction for three unrelated patients who suffered complications during treatment with PLP food supplements. – Two cases of PNPO deficiency and one case of PLP-dependent epileptic encephalopathy without a (genetic) diagnosis are reported. These patients are critically dependent on PLP for seizure control and have suffered complications due to insufficient quality of these food supplements during the course of treatment. Complications include the occurrence of seizures following the administration of suspected low quality PLP, inactive PLP due to light exposure, a PLP intoxication, resisting administration and post-administration vomiting as a result of the ingestion of large amounts of capsules per day. – This case series illustrates that the reliance on food supplements as anti-seizure therapy is not without risk. The treatment of PLP-dependent seizures exemplifies that PLP is administered as medication, thus there is a clear need for licensed vitamin products of pharmaceutical quality. • A rare subset of vitB6 responsive disorders requires PLP for seizure control. • No licensed PLP product exists, so patients rely on poorly regulated food supplements. • Serious complications occur as a result of supplement quality issues. • Patients with other vitamin-responsive IMDs are at risk of similar complications. • There is a clear need for licensed nutrient products of a pharmaceutical quality. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms of neurological disorders is necessary for the development of personalized medicine. When the diagnosis considers not only the disease symptoms, but also their molecular basis, treatments tailored to individual patients may be suggested. Vitamin-responsive neurological disorders are induced by deficiencies in vitamin-dependent processes. These deficiencies may occur due to genetic impairments of proteins whose functions are involved with the vitamins. This review considers the enzymes encoded by the DHTKD1, PDK3 and PDXK genes, whose mutations are observed in patients with Charcot–Marie–Tooth (CMT) disease. The enzymes bind or produce the coenzyme forms of vitamins B1 (thiamine diphosphate, ThDP) and B6 (pyridoxal-5′-phosphate, PLP). Alleviation of such disorders through administration of the lacking vitamin or its derivative calls for a better introduction of mechanistic knowledge to medical diagnostics and therapies. Recent data on lower levels of the vitamin B3 derivative, NAD+, in the blood of patients with CMT disease vs. control subjects are also considered in view of the NAD-dependent mechanisms of pathological axonal degeneration, suggesting the therapeutic potential of vitamin B3 in these patients. Thus, improved diagnostics of the underlying causes of CMT disease may allow patients with vitamin-responsive disease forms to benefit from the administration of the vitamins B1, B3, B6, their natural derivatives, or their pharmacological forms.

Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5′-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [14C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro , which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT , but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care. • We developed an in vitro pyridoxine responsiveness assay in fibroblasts of patients with GACR. • We identified a prevalent variant within the Dutch GACR cohort, p.(Gly353Asp). • We developed a new diagnostic laboratory pipeline to extend diagnostic practices in GACR. [ABSTRACT FROM AUTHOR]

In this study, fluorescent gold nanoclusters (AuNCs) conjugated with pyridoxal-5-phosphate (PLP) were synthesized, characterized, and used for Zn2+ fluorescence turn-on sensing. PLP was conjugated over the surface of papain-stabilized fluorescent gold nanoclusters (pap-AuNCs; λex = 380 nm, λem = 670 nm) by forming imine linkage. Due to this modification, the red color emitting pap-AuNCs changed to orange color emitting nanoclusters PLP_pap-AuNCs. The nano-assembly PLP_pap-AuNCs detect Zn2+ selectively by showing a notable fluorescence enhancement at 477 nm. Zn2+ detection with PLP_pap-AuNCs was quick and easy, with an estimated detection limit of 0.14 µM. Further, paper strips and cotton buds coated with PLP_pap-AuNCs were developed for affordable on-site visual detection of Zn2+. Finally, the detection of Zn2+ in actual environmental water samples served as validation of the usefulness of PLP_pap-AuNCs. [ABSTRACT FROM AUTHOR]

Previous epidemiological studies have focused on the association of dietary vitamin B6 or circulating pyridoxal-5′-phosphate (PLP) with colorectal cancer risk. This study aimed to investigate the vitamin B6 in relation to colorectal cancer risk combining the biomarkers of PLP, pyridoxal (PL) plus PLP, and PAr (the ratio of 4-pyridoxic acid over the sum of PLP and PL). A large-scale hospital-based case-control study was conducted in Guangdong Province, China, which included 1233 colorectal cancer cases and 1245 sex and age frequency-matched controls. Serum PLP, PL, and 4-pyridoxic acid (PA) were detected with ultra-high-performance liquid chromatography–tandem mass spectrometry. Unconditional logistic regression models were used to assess the odds ratios (ORs) and 95% confidence intervals (95% CIs). Serum PLP and the sum of PLP and PL were inversely associated with colorectal cancer risk, while PAr was positively associated with colorectal cancer risk. Comparing the highest with the lowest quartile, the adjusted OR (95% CI) was 0.26 (0.20–0.33, Ptrend < 0.001) for serum PLP, 0.51 (0.40–0.66, Ptrend < 0.001) for serum PLP plus PL, and 2.90 (2.25–3.75, Ptrend < 0.001) for PAr. Serum PLP and PAr had significantly stronger associations with colorectal cancer risk in the male group and smoking group. Our results supported the protective role of vitamin B6 in colorectal cancer risk among Chinese people. The positive association of PAr with colorectal cancer risk suggested the potential role of inflammation and oxidative stress in colorectal carcinogenesis. [ABSTRACT FROM AUTHOR]

Protolytic equilibrium constants in an aqueous solution of pyridoxal-5'-phosphate hydrazone and L-tyrosine are determined spectrophotometrically at 25.0 ± 0.1°C and near-zero ionic strength. The obtained constants agree with the literature data for similar compounds. [ABSTRACT FROM AUTHOR]

Abstract Vitamin B6‐dependent epilepsies are a heterogeneous group of disorders characterized by decreased availability of the active cofactor pyridoxal‐5′‐phosphate (PLP). While pathogenic variants in ALDH7A1 or PNPO genes account for most cases of these disorders, biallelic pathogenic variants in PLPBP have been shown to cause a form of early onset vitamin B6‐dependent epilepsy (EPVB6D). PLPBP is thought to play a role in the homeostatic regulation of vitamin B6, by supplying PLP to apoenzymes while limiting side‐reaction toxicity related to excess unbound PLP. Neonatal‐onset intractable seizures that respond to pyridoxine and/or PLP are a predominant feature of EPVB6D in humans. Unlike other causes of vitamin B6‐dependent epilepsies; however, a specific biomarker for this disorder has yet to be identified. Here we present data from a premature infant found to have pathogenic variants in PLPBP and propose that prematurity may provide an additional clue for early consideration of this diagnosis. We discuss these findings in context of previously published genotypic, phenotypic, and metabolic data from similarly affected patients.

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1–80 mM l-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s−1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s−1) value obtained by assaying at 1–50 mM l-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs. [ABSTRACT FROM AUTHOR]

The proposed mechanism for tryptophan synthase shows βLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4' for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis). ε-¹⁵N-lysine TS was prepared to access the protonation state of βLys87 using ¹⁵N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the β-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Guest Editors: Andrea Mozzarelli and Loredano Pollegioni.

Abstract Guanidinocalix[5]arene and fluorescein reporter pair has been chosen to set up a supramolecular tandem assay principle based on the differential recognition of pyridoxal‐5′‐phosphate (the substrate of alkaline phosphatase, ALP), pyridoxal (the product of ALP) and phosphate (the product of ALP). This supramolecular tandem assay system offers an opportunity to monitor the activity of ALP in a label‐free, continuous, and real‐time manner. More importantly, a calibration curve can be given for selective and quantitative detection of pyridoxal‐5′‐phosphate (biomarker for several diseases).

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background : Hypophosphatasia (HPP) is a rare metabolic bone disorder caused by mutations in the alkaline phosphatase (ALPL) gene, and characterized by low circulating alkaline phosphatase (ALP) levels and bone, muscle, dental and systemic manifestations. In this case series we investigate the clinical spectrum, genetic and biochemical profile of adult HPP patients from the University Hospitals Leuven, Belgium. Methodology : Adults with HPP were identified through medical record review. Inclusion criteria were: (1) age ≥ 16 yr; (2) consecutively low ALP levels not explained by secondary causes; (3) one or more of the following supporting criteria: biochemical evidence of elevated enzyme substrates; subtrochanteric fractures, metatarsal fractures or other typical clinical features; family history of HPP; a known or likely pathogenic ALPL mutation. Results : Nineteen patients met our inclusion criteria (n = 2 infantile, n = 6 childhood, n = 10 adult-onset HPP and one asymptomatic carrier). Fractures and dental abnormalities were the most reported symptoms. Fatigue was reported in n = 7/19 patients (37%), three of which had previously been misdiagnosed as having chronic fatigue syndrome and/or fibromyalgia. Empirical pyridoxine therapy in four patients (without seizures) did not provide symptomatic relief. N = 7/19 patients (37%) were inappropriately treated or planned to be treated with antiresorptive treatment. Two patients developed atypical femoral fractures following exposure to bisphosphonates and/or denosumab. Patients detected by screening were less severely affected, while patients with homozygous or compound heterozygous mutations had the most severe symptoms, significantly lower circulating ALP levels (p = 0.013) and significantly higher pyridoxal-5'-phosphate (p = 0.0018) and urinary phosphoethanolamine (p = 0.0001) concentrations. Conclusions : Screening may detect mainly less severely affected individuals, which may nevertheless avoid misdiagnosis and inappropriate antiresorptive drug exposure. Patients with biallelic mutations had more severe symptoms, significantly lower ALP and higher substrate levels. Whether the latter finding has implications for the classification and treatment of HPP should be investigated further in larger cohorts. [ABSTRACT FROM AUTHOR]

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 (Ld PdxK) was cloned and then purified using affinity chromatography. Ld PdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of Ld PdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed Ld PdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, Ld PdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of Ld PdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of Ld PdxK 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 Ld PdxK an attractive candidate for the designing of novel and specific inhibitors. [ABSTRACT FROM AUTHOR]

Threonine synthase catalyses the conversion of O -phospho- l -homoserine and a water molecule to l -threonine and has the most complex catalytic mechanism among the pyridoxal 5′-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The K d for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site. [ABSTRACT FROM AUTHOR]

[Display omitted] • Proposed a 31P qNMR method for simultaneous assay of active vitamins B 1 , B 2 , and B 6. • Optimized extraction and NMR conditions for multivitamin supplement analysis. • The proposed method was successfully validated following the ICH guidelines. • Applicability of the method for multivitamin supplement analysis was tested. With increasing demand for active vitamins in functional foods and supplements, accurate quantification is crucial to ensure that quality and safety standards are met. This study presents the development of a phosphorous-31 quantitative nuclear magnetic resonance (31P qNMR) spectroscopy method for the simultaneous analysis of the active forms of vitamins B 1 , B 2 , and B 6 in multivitamin supplements. The 31P NMR signals of the analytes and internal standard were effectively resolved and easily distinguishable. The method was validated in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and stability. The curves for all three vitamins exhibited a high degree of linearity, with correlation coefficients (R2) exceeding 0.999. The LOD (0.04–0.07 mg mL−1) and the LOQ (0.14–0.24 mg mL−1) were adequate for analysis of multivitamin supplements, and the accuracy (99.2–102.0%) was excellent. The relative standard deviations for repeatability (1.06–1.89%) and intermediate precision (2.51–3.25%) were considered acceptable. The developed 31P qNMR method was successfully applied to commercial multivitamin supplements, demonstrating its practicality. Therefore, the 31P qNMR method is expected to be suitable for the simultaneous quantification of active vitamins B 1 , B 2 , and B 6 and an effective analysis method for multivitamin preparations. [ABSTRACT FROM AUTHOR]

Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for ectoine synthesis and import to maintain physiologically adequate cellular ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the ectoine biosynthetic pathway.

Guanidinocalix[5]arene and fluorescein reporter pair has been chosen to set up a supramolecular tandem assay principle based on the differential recognition of pyridoxal‐5′‐phosphate (the substrate of alkaline phosphatase, ALP), pyridoxal (the product of ALP) and phosphate (the product of ALP). This supramolecular tandem assay system offers an opportunity to monitor the activity of ALP in a label‐free, continuous, and real‐time manner. More importantly, a calibration curve can be given for selective and quantitative detection of pyridoxal‐5′‐phosphate (biomarker for several diseases). [ABSTRACT FROM AUTHOR]

Background: Vitamin B-6 (B-6), in the form of pyridoxal 5'phosphate (PLP), is critical for one-carbon metabolism reactions and cellular function. Plasma PLP concentration decreases throughout pregnancy, but the functional consequences of this have not been studied. Plasma cystathionine is a sensitive indicator of suboptimal B-6 status in healthy adults.Objectives: The aim of this study was to determine the relation between plasma PLP and cystathionine concentrations, and to assess longitudinal changes in plasma concentrations of metabolites of one-carbon metabolism, including total homocysteine (tHcy), cysteine, methionine, glycine, serine, and glutathione, over the course of pregnancy.Design: This was a prospective cohort study of 186 healthy Brazilian pregnant women (20-40 y). Plasma PLP and metabolite concentrations were quantified in fasting maternal blood samples collected between 5-13, 20-26, and 30-36 weeks of gestation. Linear mixed regression models were used to determine the association of 1) first-trimester PLP tertiles, and 2) the variation of PLP concentration throughout pregnancy, with related metabolite concentrations across weeks of gestation.Results: Median (IQR) PLP concentration decreased from 36.2 (29.2-44.5) to 21.0 (15.9-26.0) to 16.8 (12.9-21.4) nmol/L in the first, second, and third trimester, respectively, whereas cystathionine concentration increased from 63.2 (49.7-78.9) to 122 (98.0-167) to 143 (114-193) nmol/L, respectively (both P < 0.001). The variation of PLP throughout pregnancy was inversely associated with cystathionine concentration across weeks of gestation, after adjusting for confounding factors; β (95% CI) = -0.387 (-0.752, -0.219), P = 0.04. This association significantly differed by trimester and was strongest in the third trimester. Plasma concentrations of glycine, serine, methionine, cysteine, and tHcy decreased, and that of glutathione increased, between the first and second trimesters (all P < 0.05).Conclusions: The variation of PLP concentration predicted cystathionine concentration throughout pregnancy. Increases in plasma cystathionine across trimesters may reflect maternal intracellular B-6 deficiency. [ABSTRACT FROM AUTHOR]

Stereospecific generation of α-amino ketones from common α-amino acids is difficult to achieve, often employing superstoichiometric alkylating reagents and requiring multiple protecting group manipulations. In contrast, the α-oxoamine synthase protein family performs this transformation stereospecifically in a single step without the need for protecting groups. Herein, we detail the characterization of the 8-amino-7-oxononanoate synthase (AONS) domain of the four-domain polyketide-like synthase SxtA, which natively mediates the formation of the ethyl ketone derivative of arginine. The function of each of the four domains is elucidated, leading to a revised proposal for the initiation of saxitoxin biosynthesis, a potent neurotoxin. We also demonstrate the synthetic potential of SxtA AONS, which is applied to the synthesis of a panel of novel α-amino ketones. 1 Introduction 2 Native SxtA Module Activity 3 New Reactions with SxtA AONS 4 Conclusions and Outlook [ABSTRACT FROM AUTHOR]

Kynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5′-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and β-benzoyl-L-alanine, but not O-benzoyl- l -serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with β-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with β-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The 1H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold reduction of k cat and 30-fold reduction in k cat / K m with L-kynurenine, but very little effect on the reaction of O-benzoyl- l -serine. Thus, the rate-determining step in the reaction of S36A Pfkynase is the C β -C γ bond cleavage. These results support the hypothesis that Ser-36 together with Tyr-226 is part of an oxyanion hole that polarizes the carbonyl of the substrate in the catalytic mechanism of Pfkynase. Unlabelled Image • Asp-132 and Asp-201 have been mutated to alanine and glutamate. • Asp-132 and Asp-201 modulate the reactivity of the PLP cofactor. • Ser-36 may donate a hydrogen bond the substrate carbonyl oxygen. • The S36A mutation results in ~1000-fold reduction in the chemical step. • Ser-36 with Tyr-225 may form an oxyanion hole in the mechanism of kynureninase. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP-binding cassette (ABC) transporters ABCB7 in humans, Atm1 in yeast and ATM3 in plants, are highly conserved in their overall architecture and particularly in their glutathione binding pocket located within the transmembrane spanning domains. These transporters have attracted interest in the last two decades based on their proposed role in connecting the mitochondrial iron–sulfur (Fe–S) cluster assembly with its cytosolic Fe–S cluster assembly (CIA) counterpart. So far, the specific compound that is transported across the membrane remains unknown. In this report we characterized the ABCB7-like transporter Rcc02305 in Rhodobacter capsulatus , which shares 47% amino acid sequence identity with its mitochondrial counterpart. The constructed interposon mutant strain in R. capsulatus displayed increased levels of intracellular reactive oxygen species without a simultaneous accumulation of the cellular iron levels. The inhibition of endogenous glutathione biosynthesis resulted in an increase of total glutathione levels in the mutant strain. Bioinformatic analysis of the amino acid sequence motifs revealed a potential aminotransferase class-V pyridoxal-5′-phosphate (PLP) binding site that overlaps with the Walker A motif within the nucleotide binding domains of the transporter. PLP is a well characterized cofactor of L-cysteine desulfurases like IscS and NFS1 which has a role in the formation of a protein-bound persulfide group within these proteins. We therefore suggest renaming the ABCB7-like transporter Rcc02305 in R. capsulatus to PexA for P LP binding ex porter. We further suggest that this ABC-transporter in R. capsulatus is involved in the formation and export of polysulfide species to the periplasm. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Aromatic amino acid transaminases are involved in several metabolic pathways. • Structures of transaminases Aro8p and Aro9p from C. albicans are determined. • The structure of Aro9p is the first determined structure of Aro9 aminotransferase. • Aromatic transaminase Aro8p structure is the first from a pathogenic fungus. • Ability to process many substrates is related to the enzymes' special architecture. Abstract Aminotransferases catalyze reversibly the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5′-phosphate (PLP) as a cofactor. Various aminotransferases acting on a range of substrates have been reported. Aromatic transaminases are able to catalyze the transamination reaction with both aromatic and acidic substrates. Two aminotransferases from C. albicans , Aro8p and Aro9p, have been identified recently, exhibiting different catalytic properties. To elucidate the multiple substrate recognition of the two enzymes we determined the crystal structures of an unliganded Ca Aro8p, a complex of Ca Aro8p with the PLP cofactor bound to a substrate, forming an external aldimine, Ca Aro9p with PLP in the form of internal aldimine, and Ca Aro9p with a mixture of ligands that have been interpreted as results of the enzymatic reaction. The crystal structures of both enzymes contains in the asymmetric unit a biologically relevant dimer of 55 kDa for Ca Aro8 and 59 kDa for Ca Aro9p protein subunits. The ability of the enzymes to process multiple substrates could be related to a feature of their architecture in which the active site resides on one subunit while the substrate-binding site is formed by a long loop extending from the other subunit of the dimeric molecule. The separation of the two functions to different chemical entities could facilitate the evolution of the substrate-binding part and allow it to be flexible without destabilizing the conservative catalytic mechanism. [ABSTRACT FROM AUTHOR]

Abstract A 2-year-old girl required medical attention for a sudden onset of repetitive tonic-clonic convulsions after ingesting 20–30 ginkgo seeds. Concentrations of the major forms of circulating vitamin B 6 , pyridoxal-5′-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid, as well as the known ginkgo seed toxin 4′- O -methylpyridoxine (MPN) were measured in the serum and cerebrospinal fluid (CSF). PLP is an active form of vitamin B 6 and necessary for γ-aminobutyric acid (GABA) production. High MPN concentrations were observed in both the serum and CSF. As the PLP to PL ratio was markedly decreased in serum and CSF examinations, we suspected the ratio to be important in GABA production. This case report provides novel information on the metabolism of vitamin B 6 in humans as a result of ginkgo seed poisoning. [ABSTRACT FROM AUTHOR]

Background: Inflammation can increase vitamin B6 uptake and catabolism. Higher vitamin B6 turnover [4-pyridoxic acid (4-PA)/pyridoxal 5'-phosphate (PLP) ratio], was associated with mortality risk in the general population., Objectives: We aimed to investigate the association between 4-PA/PLP and long-term mortality in patients with type 2 diabetes mellitus (T2DM), an inflammatory disease., Methods: In this prospective cohort study from the National Health and Nutrition Examination Survey (NHANES) cycles 2005-2010, the concentrations of 4-PA and PLP in plasma were measured using high-performance liquid chromatography, with mortality data updated to 31 December 2019. We included 2074 patients with T2DM aged between 20 and 85 y at baseline., Results: There were 739 deaths among 2279 patients with T2DM with a median follow-up of 11.83 y. In the age- and sex-adjusted COX model (model 1), 4-PA/PLP was positively associated with mortality in patients with T2DM [hazard ratio (HR) and 95% confidence interval (CI) highest compared with lowest quartiles: 35.55 (18.29, 69.09); P < 0.001], and in model 3, which was adjusted for demographics as well as inflammation, nutrition, and renal function, high 4-PA/PLP concentrations remained an independent risk factor for mortality in patients with T2DM [HR (95% CI) highest compared with lowest quartiles: 5.03 (2.46, 10.30); P < 0.001]. In restricted cubic spline (RCS), the link between 4-PA/PLP and all-cause mortality displays a positive correlation. Patients with died within the previous 2 y were excluded, the sensitivity analysis had no effect on the association between 4-PA/PLP and mortality in patients with T2DM. Finally, comparable results were found in subgroup analyses of specific-cause mortality., Conclusion: Higher vitamin B6 turnover is associated with long-term mortality risk in patients with T2DM. 4-PA/PLP may serve as a convenient prognostic marker in T2DM management., (© 2023 The Authors.)

Salmonella enterica serovar Typhimurium utilizes a wide range of growth substrates, some of which are relatively novel. One of these unusual substrates is d -glucosaminate, which is metabolized by the enzymes encoded in the dga operon. d -Glucosaminate is transported and converted to d -glucosaminate-6-phosphate (G6P) by a phosphotransferase system, composed of DgaABCD. The protein product of dga E, d -glucosaminate-6-phosphate ammonia lyase (DGL), converts G6P to 2-keto-3-deoxygluconate-6-phosphate, which undergoes a retroaldol reaction catalyzed by the DgaF protein to give d -glyceraldehyde-3-phosphate and pyruvate. We have now developed an improved synthesis of G6P which gives a higher yield. The DGL reaction is of mechanistic interest because it is one of only a few enzymes in the pyridoxal-5′-phosphate (PLP) dependent aminotransferase superfamily known to catalyze reaction of a d -amino acid substrate. The pH dependence of DGL shows an optimum at 7.5–8.5, suggesting a requirement for a catalytic base. α-Glycerophosphate and inorganic phosphate are weak competitive inhibitors, with K i values near 30 mM, and d -serine is neither a substrate nor an inhibitor. We have found in rapid-scanning stopped-flow experiments that DGL reacts rapidly with its substrate to form a quinonoid intermediate with λ max = 480 nm, within the dead time (ca. 2 msec), which then rapidly decays (k = 279 s − 1 ) to an intermediate with absorption between 330 and 350 nm, probably an aminoacrylate complex. We suggest a mechanism for DGL and propose that the unusual stereochemistry of the DGL reaction requires a catalytic base poised on the opposite face of the PLP-substrate complex from the other members of the aminotransferase superfamily. [ABSTRACT FROM AUTHOR]

STM2360 is a gene located in a small operon of undetermined function in Salmonella enterica serovar Typhimurium LT2. The amino acid sequence of STM2360 shows significant similarity (∼30% identity) to diaminopimelate decarboxylase (DapDC), a Fold III pyridoxal-5′-phosphate (PLP) dependent enzyme involved in l -lysine biosynthesis. We have found that the protein coded by STM2360 has a previously undocumented catalytic activity, d -ornithine/ d -lysine decarboxylase (DOKDC). The reaction products, cadaverine and putrescine, respectively, were identified by NMR and mass spectrometry. The substrate specificity of DOKDC is d -Lysine > d -Ornithine. This is the first pyridoxal-5′-phosphate dependent decarboxylase identified to act on d -amino acids. STM2358, located in the same operon, has ornithine racemase activity. This suggests that the physiological substrate of the decarboxylase and the operon is ornithine. Homologs of STM2360 with high sequence identity (>80%) are found in other common enterobacteria, including species of Klebsiella , Citrobacter , Vibrio and Hafnia , as well as Clostridium in the Firmicutes, and Pseudomonas . [ABSTRACT FROM AUTHOR]

Background Pyridoxal-5 ′ -phosphate oxidase (PNPO) deficiency presents as a severe neonatal encephalopathy responsive to pyridoxal-5 ′ -phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected genetic variants on PNPO gene whose pathogenic role and clinical expression remain to be established. Objective This paper aims to characterize the functional effects of the c.347G > A (p.Arg116Gln) mutation in the PNPO gene in order to define its pathogenicity and describe the clinical features of new patients with epilepsy carrying this mutation. Methods Arg116Gln protein variant was expressed as recombinant protein. The mutant protein was characterized with respect to structural and kinetic properties, thermal stability, binding constants of cofactor (FMN) and product (PLP). We also reviewed clinical data of 3 new patients carrying the mutation. Results The Arg116Gln mutation does not alter the overall enzyme structure and only slightly affects its catalytic efficiency; nevertheless, this mutation affects thermal stability of PNPO, reduces its affinity for FMN and impairs transfer of PLP to PLP-dependent enzymes. Three boys with seizure onset between 8 months and 3 years of age, carrying the Arg116Gln mutation, are described. These three patients exhibited different seizure types associated with interictal EEG abnormalities and slow background activity. Mild/moderate intellectual disability was observed in 2/3 patients. A dramatic therapeutic response to pyridoxine was observed in the only patient who still had active seizures when starting treatment, while in all three patients interictal EEG discharges and background activity improved after pyridoxine treatment was initiated. Conclusions The reported data support a pathogenic role of the c.347G > A (p.Arg116Gln) mutation in PNPO deficiency. The later onset of symptoms and the milder epilepsy phenotype of these expand the disease phenotype. [ABSTRACT FROM AUTHOR]

As our precursory stage we have focus straight forward on clean catalytic approach for the production of C3 substituted pyridoxal-5′-phosphate analogues of vitamin B 6 , and other environmental protocols like photocatalytic activity, green fossil fuels and c–c coupling using efficient biocompatible eggshell related unrivalled materials which show versatility of the catalytic effect on different inorganic support. The eggshell immobilized nanoparticles have encouraging relevance in creation of new molecules and can advantageously be studied by various spectroscopic, thermal and elemental analyses like powder X-ray diffraction (XRD), Raman spectroscopy, UV – vis, Scanning electron microscopy (SEM), Energy dispersive X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) surface area analysis. The elucidate nature of nanoparticles offer: more active site acts as lewis acid, vacancies on the catalyst surface and good to better yield of C3 substituted deoxy and 2-nor deoxy coenzyme pyridoxine (PN), coupling products propargylamines (PA), photo degrading enhancement of MB and nucleophilic substituted fatty acid (BD). This enzyme cofactor explore molecular synthons to synthetic equivalent: 3-deoxy and 2-nor-3-deoxy pyridoxal (PL), pyridoxal oxime (PO), pyridoxamine (PM) and mono phosphate derivative of 3-deoxyPM, 3-deoxyPL respectively and chemistry of selective oxidation and schiff base mechanism was studied and complemented through combined experimental and theoretical molecular orbital calculation consequently. The heterogeneous catalyst has strong selective ability towards selective reducing pyridine diester, bioactive intermediates substances and holds vast potential towards separation for the photogenerated electron-hole pairs and renewable, nontoxic, biodegradable green fossil fuels. The catalyst including environmental concern is reapplicable and strong impressive that can unfold the space of worthy metal component widely and facilitate the scope to take a vital role in different fileds like catalysis, biochemistry, nanoscience, energy and materials science. [ABSTRACT FROM AUTHOR]

To analyze the clinical and genetic characteristics of Chinese patients with pyridox(am)ine-5′-phosphate oxidase (PNPO) deficiency. The clinical presentations and the responses to treatments were analyzed in 4 patients. Blood and urinary metabolic screenings, electroencephalogram (EEG), brain magnetic resonance imaging (MRI) and epilepsy-related genes detection were performed in all patients. Patient 1 and 2 were identical twin brothers, who were born at 35 w gestation with a sign of encephalopathy. Their seizures started within the first day and could not be controlled by pyridoxine or pyridoxal-5′-phosphate (PLP) completely. Patient 3 presented seizures at 5 months, responding well to pyridoxine. Seizures in patient 4 began at 40 days after birth and were controlled by valproic acid and topiramate. EEG showed atypical hypsarrhythmia or multifocal epileptiform discharges in 3 patients, and showed normality in patient 4. MRI showed nonspecific abnormality or normality. Blood metabolic screening showed multiple amino acids level abnormalities in all cases. Urinary metabolic screening showed vanillactic acid prominently elevated in 3 patients. Genetic analysis revealed 5 mutations of PNPO, three of which were novel. The mutation c.445_448del was carried by the twins and patient 3. Assessment of psychomotor development indicated severe delay in 3 patients and borderline to mild delay in patient 3. This is the first time to report patients with PNPO deficiency diagnosed by gene analysis in China. The novel clinical characteristics and novel mutations found here expanded the phenotypes and genotypes of this disease. Further, the frameshift mutation c.445_448del might be high prevalence in PNPO deficiency in Chinese patients. [ABSTRACT FROM AUTHOR]

The isoleucine 2-epimerase from Lactobacillus buchneri has been previously identified and characterized to catalyze the pyridoxal 5′-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of nonpolar amino acids from L- to D-form and vice versa , in particular isoleucine. In this study, crystal structures of both native and PLP-complex forms of this racemase are presented at 2.6 and 2.15 Å resolution, respectively. Both structures show that the protein belongs to the fold-type I subgroup of PLP-dependent enzymes and is very close to aminobutyrate aminotransferases family, as it has been suspected because of their sequence homology. The extensive structural comparison with fold-type I enzymes with known amino acid racemization activities, including the α-amino-ε-caprolactam racemase from Achromobacter obae and the cystathionine β-lyase from Escherichia coli , allows us to identify the active site residues responsible for its nonpolar amino acid recognition and reactivity specificity. Our observations also suggest that the racemization reaction by the fold-type I racemases may generally occur thanks to a revised two-base mechanism. Lastly, both structures reveal details on the conformational changes provoked by PLP binding that suggest an induced fit of the active site “entrance door”, necessary to accommodate PLP and substrate molecules. [ABSTRACT FROM AUTHOR]