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Spectrochimica Acta. Part A, Molecular... Jun 2020In the present paper, the kinetics of a reaction between bovine serum albumin (BSA) and pyridoxal, pyridoxal 5'-phosphate was studied, apparent rate constant of product...
In the present paper, the kinetics of a reaction between bovine serum albumin (BSA) and pyridoxal, pyridoxal 5'-phosphate was studied, apparent rate constant of product formation and dissociation as well as binding constants were determined. Pyridoxal 5'-phosphate hydrazones of isonicotinic, picolinic, 2-furoic, thiophene-2-carboxylic, pyrazinoic acids binding to BSA was studied by spectrofluorimetry, stability constants of the associates were calculated from experimental data using maximal likelihood approach. The changes in the secondary structure of BSA induced by hydrazones addition were studied by IR spectroscopy. New freely available software for curve fitting was developed as a part of the software kit designed for the solution chemistry and used for a specific problem of this study, IR spectra processing.
Topics: Animals; Cattle; Hydrazines; Protein Structure, Secondary; Pyridoxal Phosphate; Serum Albumin, Bovine
PubMed: 32120288
DOI: 10.1016/j.saa.2020.118165 -
Chembiochem : a European Journal of... Nov 2019The physiological role of biogenic aldehydes, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL), has been associated with cardiovascular and neurodegenerative disorders....
The physiological role of biogenic aldehydes, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL), has been associated with cardiovascular and neurodegenerative disorders. The availability of these substrates is limited and robust synthetic methodologies would greatly facilitate further biological studies. Herein, a transaminase-mediated single-step process in continuous mode, which leads to excellent product yields (90-95 %), is reported. Coimmobilization of the pyridoxal phosphate (PLP) cofactor eliminated the need for exogenous addition of this reagent without affecting the longevity of the system, delivering a truly self-sufficient process.
Topics: Aldehydes; Amines; Bacterial Proteins; Biocatalysis; Halomonas; Pyridoxal Phosphate; Transaminases
PubMed: 31158309
DOI: 10.1002/cbic.201900356 -
Molecules (Basel, Switzerland) Oct 2022Today, complexes of gold(I) and gold(III) are recognized as promising drugs for the treatment of bacterial infectious diseases and oncological diseases, respectively. It...
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 Au 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 B 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.
Topics: Hydrazones; Gold; Pyridoxal Phosphate; Water; Phosphates
PubMed: 36364171
DOI: 10.3390/molecules27217346 -
Acta Crystallographica. Section F,... Feb 2023D-Threonine aldolase (DTA) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield...
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.
Topics: Chlamydomonas reinhardtii; Glycine Hydroxymethyltransferase; Crystallography, X-Ray; Pyridoxal Phosphate; Phosphates; Substrate Specificity
PubMed: 36748339
DOI: 10.1107/S2053230X23000304 -
SLAS Discovery : Advancing Life... Jul 2018Kynurenine aminotransferase-II (KAT-II) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that acts in the tryptophan metabolic pathway by catalyzing the transamination...
Kynurenine aminotransferase-II (KAT-II) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that acts in the tryptophan metabolic pathway by catalyzing the transamination of kynurenine into kynurenic acid (KYNA). It is one of four isoforms in the KAT family, of which it is the primary homologue responsible for KYNA production in the mammalian brain. KAT-II is targeted for inhibition as KYNA is implicated in diseases such as schizophrenia, where it is found in elevated concentrations. Previously, many different approaches have been taken to develop KAT-II inhibitors, and herein fragment-based drug design (FBDD) approaches have been exploited to provide further lead compounds that can be designed into novel inhibitors. Surface plasmon resonance (SPR) was used to screen a fragment library containing 1000 compounds, of which 41 hits were identified. These hits were further evaluated with SPR, and 18 were selected for inhibition studies. From these hits, two fragments, F6037-0164 and F0037-7280, were pursued and determined to have an IC of 524.5 (± 25.6) μM and 115.2 (± 4.5) μM, respectively. This strategy shows the viability of using FBDD in gleaning knowledge about KAT-II inhibition and generating leads for the production of KAT-II inhibitors.
Topics: Drug Design; Enzyme Inhibitors; Humans; Kynurenic Acid; Kynurenine; Pyridoxal Phosphate; Small Molecule Libraries; Surface Plasmon Resonance; Transaminases
PubMed: 29537924
DOI: 10.1177/2472555218764620 -
Mikrochimica Acta Jul 2018The authors describe the first chemiluminescence (CL) based method for determination of pyridoxal 5'-phosphate (PLP). PLP is found to generate intense CL with lucigenin...
The authors describe the first chemiluminescence (CL) based method for determination of pyridoxal 5'-phosphate (PLP). PLP is found to generate intense CL with lucigenin higher than that of the conventional lucigenin-HO system by a factor of about 9.0. This new finding is used to be in a detection method for PLP via flow injection analysis (FIA). Response is linear in the 50 nM to 200 μM PLP concentration range with a correlation coefficient of 0.998, and the detection limit (at an S/N of 3) is 6.9 nM. The assay is highly selective over various amino acids, vitamins, sugars, coenzymes and metal ions cofactors. It exhibits advantages over the commonly employed HPLC methods in that it is rapid, more economic, eco-friendly and high throughput FIA detection of PLP without the need for toxic derivatization reagents, organic solvents, and HPLC instrumentation. The method was successfully applied to the determination of PLP in (spiked) human blood samples with recoveries in the range from 96.2-101.6% with % RSD < 4.0. The new system is also employed to determine lucigenin in the linear range of 0.3 to 100.0 μM with a correlation coefficient of 0.994 and the limit of detection is 0.04 μM. Graphical abstract Schematic of the chemiluminescent assay for pyridoxal 5'-phosphate (PLP). Lucigenin-PLP demonstrates 9-fold stronger chemiluminescence intensity than the lucigenin-HO system. The detection limit of PLP is 6.9 nM. The method can detect PLP in human serum with good recoveries.
Topics: Acridines; Chemistry Techniques, Analytical; Humans; Limit of Detection; Luminescence; Pyridoxal Phosphate
PubMed: 30030633
DOI: 10.1007/s00604-018-2887-2 -
International Journal of Biological... Jun 2020The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5'-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5'-phosphate...
The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5'-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5'-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors.
Topics: Amino Acid Sequence; Catalytic Domain; Humans; Leishmania donovani; Phosphotransferases (Alcohol Group Acceptor); Protein Conformation; Pyridoxal Kinase; Pyridoxal Phosphate; Pyridoxamine; Pyridoxine; Substrate Specificity
PubMed: 32105687
DOI: 10.1016/j.ijbiomac.2020.02.257 -
Journal of Neurochemistry Apr 2022Vitamins B (thiamine) and B (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their...
Vitamins B (thiamine) and B (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg but activating the Zn -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B - and B -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in K of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased K of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.
Topics: Animals; Brain; Mammals; Phosphates; Pyridoxal Kinase; Pyridoxal Phosphate; Rats; Thiamine
PubMed: 35050500
DOI: 10.1111/jnc.15576 -
International Journal of Molecular... Dec 2022Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes....
Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.
Topics: Pyridoxine; Pyridoxaminephosphate Oxidase; Dopa Decarboxylase; Pyridoxal Phosphate; Oxidoreductases; Pyridoxal Kinase
PubMed: 36614085
DOI: 10.3390/ijms24010642 -
Journal of Inherited Metabolic Disease Jan 2020Inborn errors of metabolism cause disease because of accumulation of a metabolite before the blocked step or deficiency of an essential metabolite downstream of the...
Inborn errors of metabolism cause disease because of accumulation of a metabolite before the blocked step or deficiency of an essential metabolite downstream of the block. Treatments can be directed at reducing the levels of a toxic metabolite or correcting a metabolite deficiency. Many disorders have been treated successfully first in a single patient because we can measure the metabolites and adjust treatment to get them as close as possible to the normal range. Examples are drawn from Komrower's description of treatment of homocystinuria and the author's trials of treatment in bile acid synthesis disorders (3β-hydroxy-Δ -C -steroid dehydrogenase deficiency and Δ -3-oxosteroid 5β-reductase deficiency), neurotransmitter amine disorders (aromatic L-amino acid decarboxylase [AADC] and tyrosine hydroxylase deficiencies), and vitamin B6 disorders (pyridox(am)ine phosphate oxidase deficiency and pyridoxine-dependent epilepsy [ALDH7A1 deficiency]). Sometimes follow-up shows there are milder and more severe forms of the disease and even variable clinical manifestations but by measuring the metabolites we can adjust the treatment to get the metabolites into the normal range. Biochemical measurements are not subject to placebo effects and will also show if the disorder is improving spontaneously. The hypothesis that can then be tested for clinical outcome is whether getting metabolite(s) into a target range leads to an improvement in an outcome parameter such as abnormal liver function tests, hypokinesia, epilepsy control etc. The metabolite-guided approach to treatment is an example of personalized medicine and is a better way of determining efficacy for disorders of variable severity than a randomized controlled clinical trial.
Topics: 3-Hydroxysteroid Dehydrogenases; Administration, Oral; Bile Acids and Salts; Epilepsy; Humans; Metabolic Diseases; Pyridoxal Phosphate; Pyridoxaminephosphate Oxidase; Pyridoxine; Randomized Controlled Trials as Topic; Vitamin B 6; Vitamin B 6 Deficiency
PubMed: 31222759
DOI: 10.1002/jimd.12139