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BackgroundSphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a recently recognized inborn error of metabolism associated with steroid-resistant nephrotic syndrome as well as adrenal insufficiency and immunological, neurological, and skin manifestations. SPLIS is caused by inactivating mutations in SGPL1, encoding the pyridoxal 5'phosphate-dependent enzyme sphingosine-1-phosphate lyase, which catalyzes the final step of sphingolipid metabolism. Some SPLIS patients have undergone kidney transplantation, and others have been treated with vitamin B6 supplementation. In addition, targeted therapies including gene therapy are in preclinical development. In anticipation of clinical trials, it will be essential to characterize the full spectrum and natural history of SPLIS. We performed a retrospective analysis of 76 patients in whom the diagnosis of SPLIS was established in a proband with at least one suggestive finding and biallelic SGPL1 variants identified by molecular genetic testing. The main objective of the study was to identify factors influencing survival in SPLIS subjects.ResultsOverall survival at last report was 50%. Major influences on survival included: (1) age and organ involvement at first presentation; (2) receiving a kidney transplant, and (3) SGPL1 genotype. Among 48 SPLIS patients with nephropathy who had not received a kidney transplant, two clinical subgroups were distinguished. Of children diagnosed with SPLIS nephropathy before age one (n = 30), less than 30% were alive 2 years after diagnosis, and 17% were living at last report. Among those diagnosed at or after age one (n = 18), ~ 70% were alive 2 years after diagnosis, and 72% were living at time of last report. SPLIS patients homozygous for the SPL R222Q variant survived longer compared to patients with other genotypes. Kidney transplantation significantly extended survival outcomes.ConclusionOur results demonstrate that SPLIS is a phenotypically heterogeneous condition. We find that patients diagnosed with SPLIS nephropathy in the first year of life and patients presenting with prenatal findings represent two high-risk subgroups, whereas patients harboring the R222Q SGPL1 variant fare better than the rest. Time to progression from onset of proteinuria to end stage kidney disease varies from less than one month to five years, and kidney transplantation may be lifesaving.

The pyridoxal 5′‐phosphate binding protein (PLP‐BP) is believed to play a crucial role in PLP homeostasis, which may explain why it is found in living organisms from all kingdoms. Escherichia coli YggS is the most studied homolog, but human PLP‐BP has also attracted much attention because variants of this protein are responsible for a severe form of B6‐responsive neonatal epilepsy. Yet, how PLP‐BP is involved in PLP homeostasis, and thus what its actual function is in cellular metabolism, is entirely unknown. The present study shows that YggS binds RNA and that the strength of this interaction is modulated by PLP. A key role in RNA binding is clearly played by Lys137, an invariant residue located on a protein loop away from the PLP binding site, whose importance has been highlighted previously. The interaction with RNA is evidently conserved, since it is also observed with human PLP‐BP. The RNA binding site, which is apparently located at the entrance of the PLP‐binding site, is also evolutionarily conserved. It is therefore reasonable to assume that PLP, by defining the conformation of the protein, determines the RNA binding affinity. RNA‐seq analysis of RNA co‐purified with or captured by YggS revealed SsrA and RnpB RNAs, respectively involved in trans‐translation and tRNA maturation, as the major molecular components. This work opens up new horizons for the function of the PLP‐BP, which could be related to its interaction with RNA and modulated by PLP, and thus play a role in an as yet unknown regulatory mechanism. [ABSTRACT FROM AUTHOR]

Background: Studies on the association between serum vitamin B6 status and colorectal cancer prognosis are limited and have yielded inconsistent results. This study investigated the association of pyridoxal 5′-phosphate (PLP) and pyridoxic acid ratio (PAr) index with colorectal cancer survival. Methods: A total of 1286 colorectal cancer patients diagnosed since 2010 were selected from the Guangdong Colorectal Cancer Cohort study. Serum levels of PLP, pyridoxal, and 4-pyridoxic acid were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry. The study followed overall mortality and colorectal cancer-specific mortality until December 2023. Multivariable Cox proportional hazards regression models were applied to calculate hazard ratios (HRs) and 95% confidence intervals (95% CIs). Restricted cubic spline and stratified analysis were performed. Results: During a median follow-up of 77.36 months, 331 deaths were recorded, with 293 specifically attributed to colorectal cancer. Higher PLP levels were associated with a longer overall survival (HRQ4 vs. Q1, 0.63; 95% CI: 0.46, 0.87; p for trend = 0.008) and colorectal cancer-specific survival (HRQ4 vs. Q1, 0.62; 95% CI: 0.44, 0.87; p for trend = 0.006). Non-linear associations were observed between serum PLP and overall and colorectal cancer-specific survival (p for non-linear < 0.05). However, PAr was not significantly associated with either overall survival (HRQ4 vs. Q1, 1.03; 95% CI: 0.75, 1.41) or colorectal cancer-specific survival (HRQ4 vs. Q1, 1.01; 95% CI: 0.72, 1.42). The association between serum PLP and both overall survival and colorectal cancer-specific survival (p for interaction < 0.05) varied by alcohol drinking status. Conclusions: Higher serum PLP levels, but not PAr, may be associated with improved overall and colorectal cancer-specific survival. [ABSTRACT FROM AUTHOR]

Marginal vitamin B6 (B6) deficiency is common in various segments worldwide. In a super-aged society, sarcopenia is a major concern and has gained significant research attention focused on healthy aging. To date, the primary interventions for sarcopenia have been physical exercise therapy. Recent evidence suggests that inadequate B6 status is associated with an increased risk of sarcopenia and mortality among older adults. Our previous study showed that B6 supplementation to a marginal B6-deficient diet up-regulated the expression of various exercise-induced genes in the skeletal muscle of rodents. Notably, a supplemental B6-to-B6-deficient diet stimulates satellite cell-mediated myogenesis in rodents, mirroring the effects of physical exercise. These findings suggest the potential role of B6 as an exercise-mimetic nutrient in skeletal muscle. To test this hypothesis, we reviewed relevant literature and compared the roles of B6 and exercise in muscles. Here, we provide several pieces of evidence supporting this hypothesis and discuss the potential mechanisms behind the similarities between the effects of B6 and exercise on muscle. This research, for the first time, provides insight into the exercise-mimetic roles of B6 in skeletal muscle. [ABSTRACT FROM AUTHOR]

Vitamin B1 (thiamine pyrophosphate (TPP)) and B6 (pyridoxal 5'- phosphate (PLP)) deficiencies pose significant health risks. The current measurement method employs High-Performance Liquid Chromatography (HPLC), though, Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) is considered a more sensitive and selective analytical method. However, there is a lack of LC-MS/MS-based reference intervals. Moreover, none of the existing reference intervals are established in Danish populations. Therefore, the aim of this study was to establish a reference interval for whole blood concentrations of TPP and PLP in Danish blood donors using LC-MS/MS. Blood samples were collected from healthy Danish blood donors and analysed using the reagent kit, MassChrom® Vitamins B1 and B6 in whole blood (Chromsystems Instruments & Chemicals GmbH, Munich, Germany) for quantitative determination of both TPP and PLP concentration in whole blood, using LC-MS/MS. Reference intervals were determined with non-parametric methods as the 2.5th and 97.5th percentile and presented with 90% confidence intervals (CI). In total 120 blood donors were included. The concentrations of TTP or PLP were not statistically different between sexes just as age did not affect the concentrations, hence, combined reference intervals were employed. The resulting reference intervals are: TPP, nmol/L: 101.0 (90% CI: 96.4–108.5) − 189.0 (90% CI: 184.7–192.0) and PLP, nmol/L: 64.0 (90% CI: 60.9–66.7) − 211.8 (90% CI: 168.3–231.0). In conclusion, reference intervals for whole blood TTP and PLP in a healthy Danish population were established based on a LC-MS/MS method. Furthermore, the reference intervals were not affected by age or sex. [ABSTRACT FROM AUTHOR]

Vignette: Hypophosphatasia (HPP) is the dento-osseous disorder caused by deactivating mutation(s) of ALPL, the gene that encodes the "tissue-nonspecific" isoenzyme of alkaline phosphatase (TNSALP). In HPP, 3 natural substrates of cell-surface TNSALP accumulate extracellularly; phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5′-phosphate (PLP). Hypophosphatasemia together with elevated plasma levels of PEA, PPi, and PLP comprise its biochemical signature. PPi can inhibit mineralization and in extracellular excess can impair bone and tooth hardening and perhaps explain weak muscle. Autosomal dominant or autosomal recessive inheritance from among more than 400 mutations of ALPL largely accounts for HPP's broad-ranging severity, greatest among all skeletal diseases. Pediatric HPP spans life-threatening perinatal and infantile forms, childhood forms, and odonto-HPP selectively featuring premature loss of deciduous teeth. ALPL gene testing and TNSALP supplementation therapy have bolstered familiarity with HPP, but there are new considerations for diagnosis. Herein, diagnosis of a boy's mild childhood HPP was delayed by missteps involving his medical and dental history, physical examination, radiographic findings, and clinical laboratory studies. We review how pediatric HPP is now identified. Prompt diagnosis while appreciating the broad-ranging severity of HPP underlies the safe and effective management of this inborn-error-of-metabolism. [ABSTRACT FROM AUTHOR]

Pyridoxal 5'‐phosphate (PLP)‐dependent enzymes that catalyze γ‐replacement reactions are prevalent, yet their utilization of carbon nucleophile substrates is rare. The recent discovery of two PLP‐dependent enzymes, CndF and Fub7, has unveiled unique C−C bond forming capabilities, enabling the biocatalytic synthesis of alkyl‐ substituted pipecolic acids from O‐acetyl‐L‐homoserine and β‐keto acid or aldehyde derived enolates. This breakthrough presents fresh avenues for the biosynthesis of pipecolic acid derivatives. However, the catalytic mechanisms of these enzymes remain elusive, and a dearth of structural information hampers their extensive application. Here, we have broadened the catalytic scope of Fub7 by employing ketone‐derived enolates as carbon nucleophiles, revealing Fub7's capacity for substrate‐dependent regioselective α‐alkylation of unsymmetrical ketones. Through an integrated approach combining X‐ray crystallography, spectroscopy, mutagenesis, and computational docking studies, we offer a detailed mechanistic insight into Fub7 catalysis. Our findings elucidate the structural basis for its substrate specificity, stereoselectivity, and regioselectivity. Our work sets the stage ready for subsequent protein engineering effort aimed at expanding the synthetic utility of Fub7, potentially unlocking novel methods to access a broader array of noncanonical amino acids. [ABSTRACT FROM AUTHOR]

A case of an adult with borderline AADC deficiency symptoms is presented here. Genetic analysis revealed that the patient carries two AADC variants (NM_000790.3: c.1040G > A and c.679G > C) in compound heterozygosis, resulting in p.Arg347Gln and p.Glu227Gln amino acid alterations. While p.Arg347Gln is a known pathogenic variant, p.Glu227Gln is unknown. Combining clinical features to bioinformatic and molecular characterization of the AADC protein population of the patient (p.Arg347Gln/p.Arg347Gln homodimer, p.Glu227Gln/p.Glu227Gln homodimer, and p.Glu227Gln/p.Arg347Gln heterodimer), we determined that: i) the p.Arg347Gln/p.Arg347Gln homodimer is inactive since the alteration affects a catalytically essential structural element at the active site, ii) the p.Glu227Gln/p.Glu227Gln homodimer is as active as the wild-type AADC since the alteration occurs at the surface and does not change the chemical nature of the amino acid, and iii) the p.Glu227Gln/p.Arg347Gln heterodimer has a catalytic efficiency 75% that of the wild-type since only one of the two active sites is compromised, thus demonstrating a positive complementation. By this approach, the molecular basis for the mild presentation of the disease is provided, and the experience made can also be useful for personalized therapeutic decisions in other mild AADC deficiency patients. Interestingly, in the last few years, many previously undiagnosed or misdiagnosed patients have been identified as mild cases of AADC deficiency, expanding the phenotype of this neurotransmitter disease.

Aromatic amino acid decarboxylase is a pyridoxal 5′-phosphate-dependent enzyme responsible for the synthesis of the neurotransmitters, dopamine and serotonin. Here, by a combination of bioinformatic predictions and analyses, phosphorylation assays, spectroscopic investigations and activity measurements, we determined that Ser-193, a conserved residue located at the active site, can be phosphorylated, increasing catalytic efficiency. In order to determine the molecular basis for this functional improvement, we determined the structural and kinetic properties of the site-directed variants S193A, S193D and S193E. While S193A retains 27% of the catalytic efficiency of wild-type, the two acidic side chain variants are impaired in catalysis with efficiencies of about 0.15% with respect to the wild-type. Thus, even if located at the active site, Ser-193 is not essential for enzyme activity. We advance the idea that this residue is fundamental for the correct architecture of the active site in terms of network of interactions triggering catalysis. This role has been compared with the properties of the Ser-194 of the highly homologous enzyme histidine decarboxylase whose catalytic loop is visible in the spatial structure, allowing us to propose the validation for the effect of the phosphorylation. The effect could be interesting for AADC deficiency, a rare monogenic disease, whose broad clinical phenotype could be also related to post translational AADC modifications. • AADC can be phosphorylated at the active site residue Ser193. • Ser193 plays a role in influencing AADC activity. • Phosphorylation of AADC can contribute to the broad phenotype of AADC deficiency. [ABSTRACT FROM AUTHOR]

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

Aims: Pepper fruit is a horticultural product worldwide consumed that has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H2S during fruit ripening. Results: Our data indicate that the H2S-generating cytosolic L-cysteine desulfhydrase (LCD) and the mitochondrial D-cysteine desulfhydrase (DCD) activities are downregulated during ripening but this effect was reverted after NO treatment of fruits. Innovation and Conclusion: Using as a model the non-climacteric pepper fruits at different ripening stages and under an NO-enriched atmosphere, the activity of the H2S-generating LCD and DCD was analyzed. LCD and DCD activities were downregulated during ripening, but this effect was reverted after NO treatment of fruits. The analysis of LCD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) allowed identifying three isozymes designated CaLCD I to CaLCD III, which were differentially modulated by NO and strictly dependent on pyridoxal 5′-phosphate (PLP). In vitro analyses of green fruit samples in the presence of different compounds including NO donors, peroxynitrite (ONOO−), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) triggered an almost 100% inhibition of CaLCD II and CaLCD III. This redox adaptation process of both enzymes could be cataloged as a hormesis phenomenon. The protein tyrosine (Tyr) nitration (an NO-promoted post-translational modification) of the recombinant LCD was corroborated by immunoblot and by mass spectrometry (MS) analyses. Among the 11 Tyr residues present in this enzyme, MS of the recombinant LCD enabled us to identify that Tyr82 and Tyr254 were nitrated by ONOO−, this occurring near the active center on the enzyme, where His237 and Lys260 together with the cofactor PLP are involved. These data support the relationship between NO and H2S during pepper fruit ripening, since LCD and DCD are regulated by NO during this physiological event, and this could also be extrapolated to other plant species. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamins chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation.

Concentrations of pyridoxal 5′-phosphate (PLP) in serum and whole blood are routinely measured. The suitability of these markers in capturing vitamin B6 insufficiency is not well studied.

Various d‐amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d‐amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d‐amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d‐amino acids. While in vivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d‐amino acid other than d‐Ala and d‐Glu. Here, we report the first organism that exhibits non‐canonical d‐amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d‐Ala and d‐Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d‐amino acid transaminase (DAAT) gene from Bacillus sp. YM‐1 (MB3000/mdaat+) grew well when supplemented with certain d‐amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP‐dependent enzymes of E. coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine β‐lyase (MetC) and a negative regulator of MalT activity/cystathionine β‐lyase (MalY)—these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d‐amino acid‐metabolizing enzymes. [ABSTRACT FROM AUTHOR]

Gold(III) complexes with different ligands can provide researchers with a measure against pathogenic microorganisms with antibiotic resistance. We reported in our previous paper that the UV-Vis spectra of different protonated species of complexes formed by gold(III) and five hydrazones derived from pyridoxal 5′-phosphate are similar to each other and to the spectra of free protonated hydrazones. The present paper focuses on the reasons of the noted similarity in electron absorption spectra. The geometry of different protonated species of complexes of gold(III) and hydrazones (15 structures in total) was optimized using the density functional theory (DFT). The coordination polyhedron of gold(III) bond critical points were further studied to identify the symmetry of the gold coordination sphere and the type of interactions that hold the complex together. The UV-Vis spectra were calculated using TD DFT methods. The molecular orbitals were analyzed to interpret the calculated spectra. [ABSTRACT FROM AUTHOR]

Simple Summary: Vitamin B6 is directly or indirectly involved in many key biological metabolic processes in the body in the form of coenzyme factors, and it is able to maintain the normal progress of biological responses at very low levels, playing an important role in animal health and disease. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. Its advantage of large volume injection provides adequate sensitivity. At the same time, multi-column is used to improve the shape of the peak, and the anti-interference ability is obviously improved. The system achieved rapid detection and stability quantification of PLP, PA, and PL. This method has been successfully applied to the plasma matrix of pigs, mice, and rats, providing a brand-new method for the determination of PLP, PA, and PL. Vitamin B6 is an indispensable micronutrient in organisms and is widely distributed in blood, tissues, and organs. Changes in the content and ratio of vitamin B6 can affect the entire physiological condition of the body, so it becomes particularly important to reveal the relationship between changes in its content and disease by monitoring vitamin B6 levels in the organism. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. First, PLP, PA, and PL were extracted with plasma: 0.6 M TCA: ultrapure water = 1:2:3 (v/v/v) and then derivatized. Enrichment and preliminary separation were performed on a one-dimensional column and automatically entered into a two-dimensional column for further separation. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves were >0.99. The detection limits for PLP, PA, and PL were 0.1, 0.2, and 4 nmol/L, respectively. The results showed that the system has high loading capacity, excellent resolution, and a good peak shape. This method is expected to provide applicability for the determination of PLP, PA, and PL in pharmacological, pharmaceutical, and clinical research. [ABSTRACT FROM AUTHOR]

5‐Aminolevulinic acid synthase (ALAS) is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the first and rate‐limiting step of heme biosynthesis in α‐proteobacteria and several non‐plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C‐terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C‐terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C‐terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C‐terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel β‐sheet important to Fold‐Type I PLP‐dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C‐terminus has a homolog‐specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms. PDB Code(s): 8EIM; [ABSTRACT FROM AUTHOR]

Phosphoserine aminotransferase (PSAT) is a pyridoxal 5′-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT catalyzes the transamination of 3-phosphohydroxypyruvate to 3-phosphoserine using L-glutamate as the amino donor. Although structural studies of PSAT have been performed from archaea and humans, no structural information is available from fungi. Therefore, to elucidate the structural features of fungal PSAT, we determined the crystal structure of Saccharomyces cerevisiae PSAT (ScPSAT) at a resolution of 2.8 Å. The results demonstrated that the ScPSAT protein was dimeric in its crystal structure. Moreover, the gate-keeping loop of ScPSAT exhibited a conformation similar to that of other species. Several distinct structural features in the halide-binding and active sites of ScPSAT were compared with its homologs. Overall, this study contributes to our current understanding of PSAT by identifying the structural features of fungal PSAT for the first time. [ABSTRACT FROM AUTHOR]

The glutathione (GSH) functionalized Mn-doped ZnS quantum dots (GSH_Mn_ZnS QDs) was conjugated with pyridoxal 5'-phosphate (PLP). The –CHO group of vitamin B6 cofactor PLP interacted with the -NH2 group of GSH functionalized Mn_ZnS QDs. The conjugation of PLP quenched the fluorescence emission of GSH_Mn_ZnS QDs at 601 nm. Addition of alkaline phosphatase (ALP) catalytically dephosphorylated the PLP into pyridoxal that restored the fluorescence emission of GSH_Mn_ZnS QDs. With a sensitivity of 0.035 U/L, the PLP conjugated GSH_Mn_ZnS QDs was applied to quantify ALP activity in human serum and plasma. Further, the developed nanoprobe PLP conjugated GSH_Mn_ZnS QDs was also applied to detect Al3+. The complexation-induced fluorescence enhancement was observed at 492 nm upon the interaction of Al3+ with the PLP conjugated GSH_Mn_ZnS QDs. Without any interference from other tested metal ions, this nanoprobe can be employed to detect Al3+ down to 2.30 µM. [ABSTRACT FROM AUTHOR]

Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5′-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer–monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer–monomer binding for the variants, suggest that the proper monomer–monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients. [ABSTRACT FROM AUTHOR]

d‐Threonine aldolase (DTA) is a pyridoxal‐5′‐phosphate‐dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield the d‐form β‐hydroxy‐α‐amino acid. This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Å resolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active‐site residues. On the other hand, we speculated that some non‐conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure‐guided protein engineering studies to modify reaction selectivity. [ABSTRACT FROM AUTHOR]

Abstract Background Multivariable linear regression (MLR) models were previously used to predict serum pyridoxal 5′-phosphate (PLP) concentration, the active coenzyme form of vitamin B6, but with low predictability. We developed a deep learning algorithm (DLA) to predict serum PLP based on dietary intake, dietary supplements, and other potential predictors. Methods This cross-sectional analysis included 3778 participants aged ≥20 years in the National Health and Nutrition Examination Survey (NHANES) 2007-2010, with completed information on studied variables. Dietary intake and supplement use were assessed with two 24-hour dietary recalls. We included potential predictors for serum PLP concentration in the models, including dietary intake and supplement use, sociodemographic variables (age, sex, race-ethnicity, income, and education), lifestyle variables (smoking status and physical activity level), body mass index, medication use, blood pressure, blood lipids, glucose, and C-reactive protein. We used a 4-hidden-layer deep neural network to predict PLP concentration, with 3401 (90%) participants for training and 377 (10%) participants for test using random sampling. We obtained outputs after sending the features of the training set and conducting forward propagation. We then constructed a loss function based on the distances between outputs and labels and optimized it to find good parameters to fit the training set. We also developed a prediction model using MLR. Results After training for 105 steps with the Adam optimization method, the highest R 2 was 0.47 for the DLA and 0.18 for the MLR model in the test dataset. Similar results were observed in the sensitivity analyses after we excluded supplement-users or included only variables identified by stepwise regression models. Conclusions DLA achieved superior performance in predicting serum PLP concentration, relative to the traditional MLR model, using a nationally representative sample. As preliminary data analyses, the current study shed light on the use of DLA to understand a modifiable lifestyle factor.

The Campylobacter jejuni bacterium, which causes foodborne enteritis in humans, expresses the uncharacterized protein CJ0600. Based on sequence analysis, CJ0600 has been proposed to function as a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AccDA) or cysteine desulfhydrase (CysDS). However, it has never been investigated whether CJ0600 exerts AccDA or CysDS activity or how CJ0600 mediates its enzymatic activity. To reveal the structural features necessary for the function of CJ0600, we determined the crystal structure of CJ0600 and characterized its enzymatic activity. CJ0600 contains two domains and features an interdomain pocket, which accommodates a pyridoxal 5′-phosphate (PLP) molecule as a Schiff base with its lysine residue (K35), as observed in its structural homologs, including AccDA, CysDS, and serine deaminase (SerDA). However, unlike its structural homologs, CJ0600 exists as a monomer and exhibits unique structural features throughout its structure. Moreover, CJ0600 contains unique active site residues that are not observed in AccDA, CysDS, or SerDA. Consistently, phylogenetic analysis indicates that CJ0600 and its orthologs are evolutionarily distinct from AccDA, CysDS, and SerDA. Indeed, CJ0600 showed no CysDS or SerDA activity and extremely weak AccDA activity. These observations suggest that CJ0600 functions as a unique PLP-dependent enzyme that has not been reported. • We determined the crystal structure of the CJ0600 protein from Campylobacter jejuni. • CJ0600 consists of two domains and contains an interdomain pocket. • The pocket of CJ0600 accommodates pyridoxal 5′-phosphate as a putative active site. • CJ0600 displays unique structural, enzymatic, and phylogenetic features. [ABSTRACT FROM AUTHOR]

Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5′-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography–tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Pyridaben (PY) is a widely used organochlorine acaricide, which can be detected in the peripheral blood of pregnant women. Available evidence suggests that PY has reproductive toxicity. However, it remains uncertain whether prenatal PY exposure impacts neurobehavioral development in offspring. Here, we administered PY to pregnant mice at a dose of 0.5 and 5 mg kg−1 day−1 via gavage and observed anxiety-like behaviors in PY offspring aged five weeks. We then integrated the metabolome and transcriptome of the offspring's brain to explore the underlying mechanism. Metabolome data indicated that the vitamin B6 metabolism pathway was significantly affected, and the pyridoxal 5′-phosphate (PLP) concentration and the active form of vitamin B6 was significantly reduced. Moreover, the transcriptome data showed that both PLP generation-related Pdxk and anxiety-related Gad1 were significantly down-regulated. Meanwhile, there was a decreasing trend in the concentration of GABA in the hippocampal DG region. Next, we supplemented PLP at a dose of 20 mg kg−1 day−1 to the PY offspring via intraperitoneal injection at three weeks. We found up-regulated expression of Pdxk and Gad1 and restored anxiety-like behaviors. This study suggests that prenatal exposure to PY can disrupt vitamin B6 metabolism, reduce the concentration of PLP, down-regulate the expression levels of Pdxk and Gad1, inhibit the production of GABA, and ultimately lead to anxiety-like behaviors in offspring.

Background The generation of the active form of vitamin B-6, pyridoxal 5′-phosphate (PLP), in tissues is dependent upon riboflavin as flavin mononucleotide, but whether this interaction is important for maintaining vitamin B-6 status is unclear. Objective To investigate vitamin B-6 and riboflavin status, their metabolic interaction, and relationship with methylenetetrahydrofolate reductase (MTHFR) genotype in adulthood. Methods Data from 5612 adults aged 18–102 y were drawn from the Irish National Adult Nutrition Survey (NANS; population-based sample) and the Trinity-Ulster Department of Agriculture (TUDA) and Genovit cohorts (volunteer samples). Plasma PLP and erythrocyte glutathione reductase activation coefficient (EGRac), as a functional indicator of riboflavin, were determined. Results Older (≥65 y) compared with younger (<65 y) adults had significantly lower PLP concentrations (P  < 0.001). A stepwise decrease in plasma PLP was observed across riboflavin categories, from optimal (EGRac ≤1.26), to suboptimal (EGRac: 1.27–1.39), to deficient (EGRac ≥1.40) status, an effect most pronounced in older adults (mean ± SEM: 76.4 ± 0.9 vs 65.0 ± 1.1 vs 55.4 ± 1.2 nmol/L; P  < 0.001). In individuals with the variant MTHFR 677TT genotype combined with riboflavin deficiency, compared with non-TT (CC/CT) genotype participants with sufficient riboflavin, we observed PLP concentrations of 52.1 ± 2.9 compared with 76.8 ±0.7 nmol/L (P  < 0.001). In participants with available dietary data (i.e. NANS cohort, n = 936), PLP was associated with vitamin B-6 intake (nonstandardized regression coefficient β: 2.49; 95% CI 1.75, 3.24; P  < 0.001), supplement use (β: 81.72; 95% CI: 66.01, 97.43; P  < 0.001), fortified food (β: 12.49; 95% CI: 2.08, 22.91; P  = 0.019), and EGRac (β: –65.81; 95% CI: –99.08, –32.54; P  < 0.001), along with BMI (β: –1.81; 95% CI: –3.31, –0.30; P  = 0.019). Conclusions These results are consistent with the known metabolic dependency of PLP on flavin mononucleotide (FMN) and suggest that riboflavin may be the limiting nutrient for maintaining vitamin B-6 status, particularly in individuals with the MTHFR 677TT genotype. Randomized trials are necessary to investigate the PLP response to riboflavin intervention within the dietary range. The TUDA study and the NANS are registered at www.ClinicalTrials.gov as NCT02664584 (27 January 2016) and NCT03374748 (15 December 2017), respectively. Clinical Trial Registry details: Trinity-Ulster-Department of Agriculture (TUDA) study, ClinicalTrials.gov no. NCT02664584 (January 27th 2016); National Adult Nutrition Survey (NANS), ClinicalTrials.gov no. NCT03374748 (December 15th 2017). [ABSTRACT FROM AUTHOR]

Today, complexes of gold(I) and gold(III) are recognized as promising drugs for the treatment of bacterial infectious diseases and oncological diseases, respectively. It is of interest to broaden the area of potential use of gold(III) compounds to the pathogenic microorganism as well. The first step towards the development of new antibacterial drugs based on Au3+ complexes is the study of their stability in an aqueous solution. The present contribution reports on the investigation of gold(III) complexation with five hydrazones derived from a well-known biologically active compound, pyridoxal 5′-phosphate (one of the aldehyde forms of the B6 vitamin). The complex formation in aqueous solutions was confirmed by mass spectrometry and fluorescent spectroscopy. The stoichiometric composition of the complexes formed and their stability constants were determined using a UV–Vis titration method. The complexes are quite stable at physiological values of pH, as the speciation diagrams show. The results of the paper are helpful for further studies of gold(III) complexes interaction with biomacromolecules. [ABSTRACT FROM AUTHOR]

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. PDB Code(s): 7U9C, 7U9H, 7UAT, 7UAU, 7UAX, 7UBP, 7UBA, 7UB8 and 7UBQ; [ABSTRACT FROM AUTHOR]

Methionine γ-lyase (MGL) is a pyridoxal 5′-phosphate–dependent enzyme catalyzing γ-elimination in l -methionine. Pyridoxal 5′-phosphate–dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and β-protons were determined using deuterium-substituted l -methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine. • The kinetic mechanism of the γ-elimination reaction catalyzed by MGL was elucidated. • Process of protein-substrate interaction was analyzed by pre-steady state kinetics. • Kinetic isotope effects of α- and β-protons were determined. • Concerted reaction mechanisms of transformation between some intermediates were suggested. • Role of amino acid residues Y113 and Y58 located in the active site were specified. [ABSTRACT FROM AUTHOR]

The pyridoxal 5'-phosphate binding protein (PLP-BP) is believed to play a crucial role in PLP homeostasis, which may explain why it is found in living organisms from all kingdoms. Escherichia coli YggS is the most studied homolog, but human PLP-BP has also attracted much attention because variants of this protein are responsible for a severe form of B 6 -responsive neonatal epilepsy. Yet, how PLP-BP is involved in PLP homeostasis, and thus what its actual function is in cellular metabolism, is entirely unknown. The present study shows that YggS binds RNA and that the strength of this interaction is modulated by PLP. A key role in RNA binding is clearly played by Lys137, an invariant residue located on a protein loop away from the PLP binding site, whose importance has been highlighted previously. The interaction with RNA is evidently conserved, since it is also observed with human PLP-BP. The RNA binding site, which is apparently located at the entrance of the PLP-binding site, is also evolutionarily conserved. It is therefore reasonable to assume that PLP, by defining the conformation of the protein, determines the RNA binding affinity. RNA-seq analysis of RNA co-purified with or captured by YggS revealed SsrA and RnpB RNAs, respectively involved in trans-translation and tRNA maturation, as the major molecular components. This work opens up new horizons for the function of the PLP-BP, which could be related to its interaction with RNA and modulated by PLP, and thus play a role in an as yet unknown regulatory mechanism., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Enzymology, the study of enzyme structures and reaction mechanisms can be considered a classical discipline. However, enzymes cannot be freely designed to catalyze desired reactions yet, and enzymology is by no means a complete science. I have long studied the reaction mechanisms of enzymes related to amino acid metabolism, such as aminotransferases and racemases, which depend on pyridoxal 5'-phosphate, a coenzyme form of vitamin B6. During these studies, I have often been reminded that enzymatic reactions are extremely sophisticated processes based on chemical principles and enzyme structures, and have often been amazed at the evolutionary mechanisms that bestowed them with such structures. In this review, I described the reaction mechanism of various pyridoxal enzymes especially related to d -amino acids metabolism, whose roles in mammals have recently attracted attention. I hope to convey some of the significance and interest in enzymology through this review. [ABSTRACT FROM AUTHOR]

The evidence on the relationship of pyridoxal 5′-phosphate (PLP) with sleep-related problems is limited and controversial. Notably, there is a lack of studies on the general population and studies of the dose–response relationship. Therefore, we conducted a cross-sectional study to examine the associations between serum PLP concentration and sleep-related problems (sleep quality and sleep duration) in adults, using the data of the National Health and Nutrition Examination Survey 2005–2010. High-performance liquid chromatography (HPLC) was used to test PLP in blood samples. Sleep quality and sleep duration were based on self-reported data, with sleep quality categorized as sleep disorder, trouble falling asleep, waking up during the night, and daytime sleepiness. The primary analyses utilized logistic regression models and restricted cubic spline. Compared with the first quartile (Q1), the odds ratios (ORs) and 95% confidence intervals (CIs) of daytime sleepiness for the Q2 and Q3 of serum PLP concentrations were 0.76 (0.59–0.99) and 0.78 (0.62–0.98), respectively. The relationship was only significant for males. Furthermore, a non-linear dose–response relationship was observed between serum PLP concentration and the risk of daytime sleepiness. Compared with the normal sleep duration group, serum PLP concentrations were negatively associated with the risks of very short, short, and long sleep duration, with relative risk ratios (RRRs) of 0.58 (0.43–0.81) (Q4), 0.71 (0.61–0.83) (Q4) and 0.62 (0.34–0.94) (Q3), respectively. The average serum PLP concentrations were higher in people with normal sleep duration, suggesting a non-linear dose–response relationship. Our study indicated that serum PLP concentrations were negatively associated with daytime sleepiness, and this association may only exist in males. Moreover, it was also inversely related to abnormal sleep duration (very short, short, long) compared to normal sleep duration. [ABSTRACT FROM AUTHOR]

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family. [ABSTRACT FROM AUTHOR]

5-Aminolevulinate synthase (ALAS; E.C. 2.3.1.37) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the key regulatory step of porphyrin biosynthesis in metazoa, fungi, and α-proteobacteria. ALAS is evolutionarily related to transaminases and is therefore classified as a fold type I PLP-dependent enzyme. As an enzyme controlling the key committed and rate-determining step of a crucial biochemical pathway ALAS is ideally positioned to be subject to allosteric feedback inhibition. Extensive kinetic and mutational studies demonstrated that the overall enzyme reaction is limited by subtle conformational changes of a hairpin loop gating the active site. These findings, coupled with structural information, facilitated early prediction of allosteric regulation of activity via an extended C-terminal tail unique to eukaryotic forms of the enzyme. This prediction was subsequently supported by the discoveries that mutations in the extended C-terminus of the erythroid ALAS isoform (ALAS2) cause a metabolic disorder known as X-linked protoporphyria not by diminishing activity, but by enhancing it. Furthermore, kinetic, structural, and molecular modeling studies demonstrated that the extended C-terminal tail controls the catalytic rate by modulating conformational flexibility of the active site loop. However, the precise identity of any such molecule remains to be defined. Here we discuss the most plausible allosteric regulators of ALAS activity based on divergences in AlphaFold-predicted ALAS structures and suggest how the mystery of the mechanism whereby the extended C-terminus of mammalian ALASs allosterically controls the rate of porphyrin biosynthesis might be unraveled.

Vitamin B6 is an indispensable micronutrient in organisms and is widely distributed in blood, tissues, and organs. Changes in the content and ratio of vitamin B6 can affect the entire physiological condition of the body, so it becomes particularly important to reveal the relationship between changes in its content and disease by monitoring vitamin B6 levels in the organism. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. First, PLP, PA, and PL were extracted with plasma: 0.6 M TCA: ultrapure water = 1:2:3 (v/v/v) and then derivatized. Enrichment and preliminary separation were performed on a one-dimensional column and automatically entered into a two-dimensional column for further separation. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves were >0.99. The detection limits for PLP, PA, and PL were 0.1, 0.2, and 4 nmol/L, respectively. The results showed that the system has high loading capacity, excellent resolution, and a good peak shape. This method is expected to provide applicability for the determination of PLP, PA, and PL in pharmacological, pharmaceutical, and clinical research.

5-ALA is the precursor of all tetrapyrroles. 5-Aminolevulinate synthase (ALAS) catalyzes the production of 5-aminolevulinic acid (5-ALA) from glycine and succinyl-CoA. HemA from Rhodopseudomonas palustris (Rp-HemA) was reported to be a highly active ALAS. To understand the catalytic mechanism of Rp-HemA, the 2.05 Å resolution crystal structure of Rp-HemA was solved. Open, half close and close conformations were observed in the substrate-free structures. Structure comparison and sequence alignment suggest the newly observed half close conformation may also be conserved in ALAS family. The pre-existed close and half close conformations in Rp-HemA may play a key role for its high activity. • Half close conformation was observed for the first time in ALAS. • Substrate-free ALAS may adopt three conformations. • The pre-existed close and half close conformations may play a key role for the high activity. [ABSTRACT FROM AUTHOR]

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5’-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy. [ABSTRACT FROM AUTHOR]

Vitamin B6, B12 and folate are required for energy metabolism and have been identified as nutrients of concern for certain population groups. This study examined the cross-sectional association between the consumption of dairy (total dairy, milk, yogurt and cheese) and biomarkers and adequacy for these nutrients in a nationally representative sample. Twenty-four-hour dietary recall data and concentrations of RBC folate (ng/mL), serum folate (ng/mL), and serum vitamins B6 (nmol/L) and B12 (pg/mL) were obtained from the National Health and Nutrition Examination Survey 2001–2018 (n = 72,831) and were analyzed by linear and logistic regression after adjusting for demographic variables. Significance was set at p < 0.01. Mean intakes of total dairy were 2.21, 2.17, 1.83 and 1.51 cups eq among consumers aged 2–8, 9–18, 19–50 and 51+ years, respectively. Higher intakes of total dairy as well as individual dairy foods (especially milk and yogurt) were positively associated with serum and RBC folate, serum vitamin B6 and serum B12, and generally, with 9–57% lower risk of inadequate or deficient levels of these vitamins. These findings suggest that encouraging dairy consumption may be an effective strategy for improving micronutrient status and provide continued evidence to support the current dietary recommendations for dairy and dairy products. [ABSTRACT FROM AUTHOR]

Background: Multivariable linear regression (MLR) models were previously used to predict serum pyridoxal 5'-phosphate (PLP) concentration, the active coenzyme form of vitamin B6, but with low predictability. We developed a deep learning algorithm (DLA) to predict serum PLP based on dietary intake, dietary supplements, and other potential predictors.Methods: This cross-sectional analysis included 3778 participants aged ≥20 years in the National Health and Nutrition Examination Survey (NHANES) 2007-2010, with completed information on studied variables. Dietary intake and supplement use were assessed with two 24-hour dietary recalls. We included potential predictors for serum PLP concentration in the models, including dietary intake and supplement use, sociodemographic variables (age, sex, race-ethnicity, income, and education), lifestyle variables (smoking status and physical activity level), body mass index, medication use, blood pressure, blood lipids, glucose, and C-reactive protein. We used a 4-hidden-layer deep neural network to predict PLP concentration, with 3401 (90%) participants for training and 377 (10%) participants for test using random sampling. We obtained outputs after sending the features of the training set and conducting forward propagation. We then constructed a loss function based on the distances between outputs and labels and optimized it to find good parameters to fit the training set. We also developed a prediction model using MLR.Results: After training for 105 steps with the Adam optimization method, the highest R2 was 0.47 for the DLA and 0.18 for the MLR model in the test dataset. Similar results were observed in the sensitivity analyses after we excluded supplement-users or included only variables identified by stepwise regression models.Conclusions: DLA achieved superior performance in predicting serum PLP concentration, relative to the traditional MLR model, using a nationally representative sample. As preliminary data analyses, the current study shed light on the use of DLA to understand a modifiable lifestyle factor. [ABSTRACT FROM AUTHOR]

Previous evidence suggests a potential dual impact of aging and vitamin B6 (B6) deficiency on polyunsaturated fatty acid (PUFA) metabolism; gender may influence PUFA biosynthesis. Perturbation of PUFA compositions during B6 deficiency could be linked to age-related health outcomes. However, little is known about the interrelationships between vitamin B6, PUFA, and gender in the older population. Therefore, we investigated whether gender-specific associations of B6 intake and plasma pyridoxal 5'-phosphate (PLP) concentration, respectively, with plasma PUFA concentrations and ratios (eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), EPA + DHA, EPA/AA, and (EPA + DHA)/AA) existed in older adults. We further examined the relationships of adequate B6 status (PLP ≥ 20 nmol/L) with high (above median) plasma PUFA relative to deficient B6 status. This cross-sectional study analyzed 461 participants aged ≥60 years from NHANES 2003–2004. Nutrient intakes were assessed using two 24-h recalls and supplement questionnaires. PLP and PUFA concentrations were measured. Multivariate linear regression assessed the association of B6 intake and PLP with PUFA; multivariate logistic regression evaluated the relationship of adequate B6 status with high plasma PUFA, adjusting for demographic, socioeconomic, and dietary factors; physical activity; smoking; alcohol; medication; and BMI. There were interactions between gender and B6 intake on EPA (P-interaction = 0.008) and AA (P-interaction = 0.004) only, whereas no interaction existed between gender and PLP on PUFA. PLP was directly associated with EPA (β = 0.181, P = 0.002), DHA (β = 0.109, P = 0.005), EPA + DHA (β = 0.14, P = 0.002), EPA/AA (β = 0.186, P = 0.004), and (EPA + DHA)/AA (β = 0.13, P = 0.026). The odds of having high plasma EPA (adjusted (a) OR: 2.03, P = 0.049) and EPA/AA (aOR: 3.83, P < 0.0001) were greater in those with adequate B6 status compared to those with deficient B6 status. In conclusion, in US older adults, a higher PLP level was associated with a greater level of EPA, DHA, EPA + DHA, EPA/AA, and (EPA + DHA)/AA. Adequate B6 status was associated with high EPA and EPA/AA status. These findings suggest that sufficient vitamin B6 status may positively influence PUFA metabolism in older adults. [ABSTRACT FROM AUTHOR]

Pyridoxal 5′-phosphate (PLP) is the coenzyme of more than 140 enzymes and is widely used in various fields. In this study, to enhance the production of PLP in Escherichia coli BL21, the recombinant strain E. coli BL21/pETDuet-1-pdxj-zwf-dxs was constructed. The concentration of PLP in this strain was 82.69 mg/L, which was increased by 1.38-fold as compared to that in E. coli BL21. Glucose, yeast extract, and pH had an obvious impact on the concentration of PLP, and their optimal levels were 34.89 g/L, 31.17 g/L, and 10.07, respectively. The concentration of PLP under the optimal condition reached 2.23 g/L. The time-course analysis showed that the highest concentration of PLP was 2.32 g/L in recombinant strain after the induction for 12 h by 0.1 mM IPTG in a 1 L shake flask, which was increased by 38.76-fold as compared to that in E. coli BL21. This study provides a good basis for the efficient production of PLP in E. coli BL21. [ABSTRACT FROM AUTHOR]

l- Methionine γ-lyse (MGL), a pyridoxal 5′-phosphate-dependent enzyme, catalyzes the α,γ-elimination of l- methionine (l -Met) and l- homocysteine (l -Hcy) to produce α-keto acids, thiols, and ammonia. Previously, various mutant enzymes of Pseudomonas putida MGL (PpMGL) were prepared to identify a homocysteine (Hcy)-specific enzyme that would assist the diagnosis of homocystinuria. Among the mutat enzymes the Q349S mutant exhibited high degradation activity toward l- Hcy. In the present study, PpMGL Q349S was characterized; the results suggested that it could be applied to determine the amount of l -Hcy. Compared to the wild-type PpMGL, specific activities of the Q349S mutant with l -Hcy and l -Met were 1.5 and 0.7 times, respectively. Additionally, we confirmed that l -Hcy in plasma samples could be accurately detected using the Q349S mutant by preincubating it with cysteine desulfurase (CsdA). Furthermore, we determined the X-ray crystal structure of PpMGL Q349S l -Met or l -Hcy complexes Michaelis complex, germinal diamine, and external aldimine at 2.25–2.40 Å. These 3D structures showed that the interaction partner of the β-hydroxyl group of Thr355 in the wild-type PpMGL was changed to the carboxyl group of the Hcy-PLP external aldimine in the Q349S mutant. The interaction of Ser349 and Arg375 was different between l -Met and l -Hcy recognition, indicating that it was important for the recognition of the carboxyl group of the substrate. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel concept of demonstrated pyridoxal phosphate permeability in E.coli • Pyridoxal phosphate inclusion in media increases protein yield and enzymatic activity • A novel protein model system for pyridoxal phosphate linked permeation studies. PLP (Pyridoxal 5′-phosphate) dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetylserine sulphdrylase (OASS) of H.contortus and determined that the lysine-47 is very important for the binding of PLP. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli. Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01 mM (Group II), 0.025 mM (Group III), 0.05 mM (Group IV) and 0.1 mM (Group V)]. There was approximately 4.2 %, 7.2 %, 10.5 % and 18 % increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I. We could find a PLP concentration-dependent increase in expression and catalytic activity signifying the probable transport of PLP across the membrane. E.coli does not secrete any dephosphorylation factor for PLP in the immediate microenvironment. There was a significant reduction in levels of inorganic phosphate after PLP dephosphorylation in induced group in comparison to uninduced E.coli and BL21 groups, signifying PLP internalization in E.coli. The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies. [ABSTRACT FROM AUTHOR]

The reaction pyridoxal 5′-phosphate with resorcinols in EtOH in the presence of concentrated HCl gives hydrochlorides of the corresponding 4-(diarylmethyl)pyridines. Refluxing ethanol solutions of the latter leads to intramolecular dehydration giving 5H-chromeno [2,3-c]-pyridines of betaine nature. This cyclization proceeds with the participation of phenolic hydroxy groups of pyridoxal 5′-phosphate and one hydroxy group of the resorcinol moieties of these substrates. [ABSTRACT FROM AUTHOR]

Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5′-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions. [ABSTRACT FROM AUTHOR]

Cadaverine is a fundamental C5 building block in the production of polyamides. Due to the limited regeneration efficiency of intracellular pyridoxal 5'-phosphate (PLP), the current fermentation-based production of cadaverine exhibits low efficiency. In this study, we developed an Escherichia coli strain L01 by introducing lysine decarboxylase (lysine decarboxylase, LDC, a key enzyme in the synthesis of cadaverine) into a lysine-producing strain E . coli LY-4, achieving a cadaverine tier of 1.07 g/L in shake flask fermentation. Subsequently, a dual metabolic pathway enhancement strategy was proposed to synergistically strengthen both endogenous and exogenous PLP synthesis modules, thereby improving intracellular PLP synthesis. The optimized strain L11 achieved a cadaverine titer of 9.23 g/L in shake flask fermentation. Finally, the fermentation process for cadaverine production by strain L11 was optimized in a 5 L fermenter. After 48 h of fed-batch fermentation, the engineered strain L11 achieved the cadaverine titer, yield, and productivity of 54.43 g/L, 0.22 g/g, and 1.13 g/(L·h), respectively. This study provides a theoretical and technical foundation for establishing microbial cell factories for bioamine production.

YggS is a pyridoxal 5′‐phosphate (PLP)‐binding protein of the conserved COG0325 family. Despite a connection with vitamin B6 homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP‐dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP‐dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5′‐phosphate and α‐keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B6 vitamers via transamination. [ABSTRACT FROM AUTHOR]