The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.

• MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Lincosamides * chemistry   biosynthesis   metabolism   MeSH
• Pyridoxal Phosphate * metabolism   chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Publication type
• Journal Article MeSH

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

• MeSH
• Biomarkers MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Neoplasms * epidemiology   MeSH
• Prospective Studies MeSH
• Pyridoxal Phosphate MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Vitamin B 6 * MeSH
• Nutrition Surveys MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Administration, Oral MeSH
• Blood Chemical Analysis methods   MeSH
• Adult MeSH
• Calibration MeSH
• Humans MeSH
• Pyridoxal Phosphate blood   MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Thiamine administration & dosage   blood   metabolism   MeSH
• Thiamine Monophosphate blood   MeSH
• Thiamine Pyrophosphate blood   MeSH
• Vitamin B 6 blood   metabolism   MeSH
• Chromatography, High Pressure Liquid instrumentation   methods   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Pyridoxin dependentní epilepsie je autozomálně recesivně dědičné onemocnění, které se prenatálně, neonatálně a v časném dětství do 3 let projevuje farmakorezistentními epileptickými záchvaty. Jde o dědičné poruchy metabolizmu pyridoxinu asociované s mutacemi v genech ALDH7A1 nebo ALDH4A1. Pyridoxin dependentní epilepsie jsou úspěšně léčitelné vysokými dávkami pyridoxinu. Diagnostika je založena na genetickém a biochemickém vyšetření. Prezentovány jsou kazuistiky tři pacientů s typickým průběhem pyridoxin dependentní epilepsie a geneticky potvrzenou mutací v ALDH7A1 genu.

Pyridoxine-dependent epilepsy is a rare autosomal recessive hereditary disorder causing severe intractable epileptic seizures presenting typically in prenatal and neonatal period, rarely in early infancy (age up to 3 years). Pyridoxine-dependent epilepsy, caused by metabolic disturbance of pyridoxine, is associated with mutations in the ALDH7A1 or ALDH4A1 gene. Pyridoxine-dependent epilepsy is successfully treatable using high doses of pyridoxine. The diagnosis is based on biochemical and genetic examinations. Three case reports of patients with a typical clinical course of pyridoxine-dependent epilepsy and genetically detected mutation in the ALDH7A1 gene are presented.

• Keywords
• pyridoxin dependentní epilepsie, pyridoxin dependentní záchvaty, gen ALDH7A1,
• MeSH
• Aldehyde Dehydrogenase * genetics   MeSH
• Epilepsy * etiology   drug therapy   genetics   MeSH
• Genetic Diseases, Inborn MeSH
• Genetic Testing MeSH
• Humans MeSH
• Mutation MeSH
• Vitamin B 6 Deficiency MeSH
• Infant, Newborn MeSH
• Pyridoxal Phosphate deficiency   MeSH
• Pyridoxine * metabolism   deficiency   therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Publication type
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH

Role vitaminu B6 (pyridoxinu) je pro normální rozvoj a funkci centrální nervové soustavy neoddiskutovatelná. Podobně jako u dalších vitaminů skupiny B je nedostatek pyridoxinu vzácný. K funkčnímu deficitu vitaminu B6 mohou vést některé specifické genetické metabolické vady. Tento deficit je hlavní příčinou pyridoxin dependentní epilepsie u dětí. Cílem předkládaného článku je představit roli vitaminu B6 u epileptických křečí v pediatrii se zaměřením na dávkování a dostupnost léčivých přípravků s pyridoxinem, a to v různých lékových formách. Dávkování pyridoxinu je v dané indikaci přísně individuální včetně nutnosti podávat vysoké dávky až do maxima 1 g/den perorálně. Zejména pro dětskou populaci lze u vysokodávkových schémat pyridoxinu přistoupit k individuální přípravě tekutých lékových forem namísto drcení velkého množství tablet. U některých typů křečí je úspěšná i terapie aktivním metabolitem pyridoxal-5’-fosfátem.

xVitamin B6 (pyridoxine) plays a pivotal role in development and functioning of central nervous system. As with other vitamins of the B group, pyridoxine deficiency is rare, but it may occur as a result of some specific genetic disorders of metabolism. This may lead into functional deficiency of vitamin B6 and devolve into pyridoxine-dependent epilepsy in children. Pyridoxin dosing in this indication is based strictly on individual patient’s response and when reeded doses as high as 1g daily are administered orally. Especially with high dose pyridoxine regimens in paediatric population, it is preferable to avoid crushing tablets for administration and choose a suitable concentration of extemporaneously prepared oral liquid forms. In some types of neonatal and infantile seazures, therapeutic trial with pyridoxal-5’-phosphate is more appropriate choice yielding more successful results.

• Keywords
• syndrom hereditární pyridoxinové závislosti, pyridoxin dependentní epilepsie,
• MeSH
• Child MeSH
• Epilepsy * etiology   MeSH
• Infant MeSH
• Dosage Forms MeSH
• Humans MeSH
• Vitamin B 6 Deficiency drug therapy   MeSH
• Infant, Newborn MeSH
• Drug Compounding methods   MeSH
• Vitamin B 6 * administration & dosage   metabolism   adverse effects   therapeutic use   MeSH
• Recommended Dietary Allowances MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Infant, Newborn MeSH

A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

• MeSH
• Actinobacillus pleuropneumoniae metabolism   MeSH
• Adenylate Cyclase Toxin antagonists & inhibitors   chemistry   metabolism   MeSH
• Bacterial Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Bordetella pertussis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Hemolysis * MeSH
• Hemolysin Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Hexokinase MeSH
• Cells, Cultured MeSH
• Lipid Bilayers metabolism   MeSH
• Macrophages MeSH
• Mice MeSH
• Osmotic Pressure MeSH
• Cell Membrane Permeability MeSH
• Pyridoxal Phosphate analogs & derivatives   MeSH
• Rosaniline Dyes MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

• MeSH
• Blood Chemical Analysis methods   standards   MeSH
• Chromatography, Liquid MeSH
• Cystathionine beta-Synthase deficiency   metabolism   MeSH
• Homocystinuria blood   diagnosis   enzymology   MeSH
• Immunoenzyme Techniques methods   standards   MeSH
• Calibration MeSH
• Plasma chemistry   enzymology   metabolism   MeSH
• Humans MeSH
• Pyridoxal Phosphate pharmacology   MeSH
• S-Adenosylmethionine pharmacology   MeSH
• Enzyme Stability MeSH
• Case-Control Studies MeSH
• Tandem Mass Spectrometry methods   standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH

Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer's disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR's promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structurally and functionally important residues. In particular, we identify S84 and P111 as residues crucial for hSR activity and C217 and K221 as residues important for binding of the Mg2+ cofactor as well as for overall stability of the enzyme.

• MeSH
• Enzyme Activation MeSH
• Amyotrophic Lateral Sclerosis diagnosis   therapy   MeSH
• Transcription, Genetic MeSH
• Mutagenesis MeSH
• Cell Proliferation MeSH
• Pyridoxal Phosphate analogs & derivatives   MeSH
• Receptors, N-Methyl-D-Aspartate MeSH
• Serine antagonists & inhibitors   MeSH

Pyridoxin-dependentní epilepsie je autozomálně recesivně dědičné onemocnění, které se prenatálně, neonatálně a v časném dětství do 3 let projevuje farmakorezistentními epileptickými záchvaty. Jde o dědičné poruchy metabolizmu pyridoxinu asociované s mutacemi v genech ALDH7A1 nebo ALDH4A1. Podobným onemocněním je pyridoxal-fosfát dependentní epilepsie (neonatální epileptická encefalopatie) podmíněná mutacemi v PNPO genu. Pyridoxin-dependentní epilepsie jsou úspěšně léčitelné vysokými dávkami pyridoxinu. Pyridoxal-fosfát dependentní epilepsie jsou na terapii pyridoxinem refrakterní, ale reagují na léčbu pyridoxal-fosfátem. Diagnostika obou jednotek je založena na genetickém a biochemickém vyšetření.

Pyridoxine dependent epilepsy is a rare autosomal recessive hereditary disorder causing a severe intractable epileptic seizures presenting typically in prenatal and neonatal period, rarely in early infancy (age up to 3 years). Pyridoxine dependent epilepsy, caused by metabolic disturbance of pyridoxine, is associated with mutations in ALDH7A1 or ALDH4A1 gene. Similar condition, pyridoxal-phosphate dependent epilepsy (also called neonatal epileptic encephalopathy), is caused by mutations in PNPO gene. Pyridoxine dependent epilepsy is successfully treatable using high doses of pyridoxine. Neonatal epileptic encephalopathy is refractory to pyridoxine administration, however responses to treatment with pyridoxal-phosphate. The diagnosis of both pyridoxine dependent epilepsy and neonatal epileptic encephalopathy is based on biochemical and genetic examinations.

• Keywords
• pyridoxal-fosfát, neonatální epileptická encefalopatie,
• MeSH
• Aldehyde Dehydrogenase genetics   MeSH
• Molecular Diagnostic Techniques methods   utilization   MeSH
• Child MeSH
• Electroencephalography utilization   MeSH
• Humans MeSH
• Infant, Newborn, Diseases genetics   metabolism   MeSH
• Infant, Newborn MeSH
• Pyridoxal Phosphate administration & dosage   metabolism   MeSH
• Pyridoxine administration & dosage   metabolism   MeSH
• Vitamin B 6 administration & dosage   adverse effects   MeSH
• Seizures diagnosis   drug therapy   genetics   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Review MeSH

Coronary artery bypass graft (CABG) surgery is frequently performed and effective; however, perioperative complications related to ischemia-reperfusion injury, including myocardial infarction (MI), remain common and result in significant morbidity and mortality. MC-1, a naturally occurring pyridoxine metabolite and purinergic receptor antagonist, prevents cellular calcium overload and may reduce ischemia-reperfusion injury. Phase 2 trial data suggest that MC-1 may reduce death or MI in high-risk patients undergoing CABG surgery. OBJECTIVE: To assess the efficacy and safety of MC-1 administered immediately before and for 30 days after surgery in patients undergoing CABG surgery. DESIGN, SETTING, AND PARTICIPANTS: The MC-1 to Eliminate Necrosis and Damage in Coronary Artery Bypass Graft Surgery II Trial, a phase 3, multicenter, randomized, double-blind, placebo-controlled trial, with 3023 intermediate- to high-risk patients undergoing CABG surgery with cardiopulmonary bypass enrolled between October 2006 and September 2007 at 130 sites in Canada, the United States, and Germany. INTERVENTIONS: Patients received either MC-1, 250 mg/d (n = 1519), or matching placebo (n = 1504) immediately before and for 30 days after CABG surgery. MAIN OUTCOME MEASURES: The primary efficacy outcome was cardiovascular death or nonfatal MI, defined as a creatine kinase (CK) MB fraction of at least 100 ng/mL or new Q waves through postoperative day 30. RESULTS: The primary efficacy outcome occurred in 140 of 1510 patients (9.3%) in the MC-1 group and 133 of 1486 patients (9.0%) in the placebo group (risk ratio, 1.04; 95% confidence interval, 0.83-1.30; P = .76). All-cause mortality was higher among patients assigned to MC-1 than placebo at 4 days (1.0% vs 0.3%; P = .03) but was similar at 30 days (1.9% vs 1.5%; P = .44). There was no difference in the 8- to 24-hour CK-MB area under the curve between the MC-1 and placebo groups (median, 270 [interquartile range, 175-492] vs 268 [interquartile range, 170-456] hours x ng/mL; P = .11). CONCLUSION: In this population of intermediate- to high-risk patients undergoing CABG surgery, MC-1 did not reduce the composite of cardiovascular death or nonfatal MI. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00402506

• MeSH
• Purinergic P2 Receptor Antagonists MeSH
• Calcium Channel Blockers administration & dosage   therapeutic use   MeSH
• Double-Blind Method MeSH
• Myocardial Infarction etiology   prevention & control   MeSH
• Cardiopulmonary Bypass MeSH
• Coronary Artery Bypass mortality   adverse effects   MeSH
• Middle Aged MeSH
• Humans MeSH
• Receptors, Purinergic P2 MeSH
• Pyridoxal Phosphate administration & dosage   therapeutic use   MeSH
• Myocardial Reperfusion Injury etiology   prevention & control   MeSH
• Risk MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Abstracts MeSH