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Molecules (Basel, Switzerland) Mar 2020In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from (TcPanD) was selected for protein engineering to efficiently produce...
In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from (TcPanD) was selected for protein engineering to efficiently produce β-alanine. A mutant PanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (), 50 °C, and 7.0, respectively. The 1mM of Na, Ni, Co, K, and Ca stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni and Na could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of β-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.
Topics: Animals; Escherichia coli; Glutamate Decarboxylase; Kinetics; Protein Engineering; Pyridoxal Phosphate; Tribolium; beta-Alanine
PubMed: 32178239
DOI: 10.3390/molecules25061280 -
G3 (Bethesda, Md.) Feb 2019Pyridoxal 5'-phosphate (the active form of vitamin B) is a cofactor that is important for a broad number of biochemical reactions and is essential for all forms of life....
Pyridoxal 5'-phosphate (the active form of vitamin B) is a cofactor that is important for a broad number of biochemical reactions and is essential for all forms of life. Organisms that can synthesize pyridoxal 5'-phosphate use either the deoxyxylulose phosphate-dependent or -independent pathway, the latter is encoded by a two-component pyridoxal 5'-phosphate synthase. contains three paralogs of the two-component pyridoxal 5'-phosphate synthase. Past work identified the biochemical activity of Snz1p, Sno1p and provided data that was involved in pyridoxal 5'-phosphate biosynthesis. Snz2p and Snz3p were considered redundant isozymes and no growth condition requiring their activity was reported. Genetic data herein showed that either or are required for efficient thiamine biosynthesis in Further, or alone could satisfy the cellular requirement for pyridoxal 5'-phosphate (and thiamine), while was sufficient for pyridoxal 5'-phosphate synthesis only if thiamine was provided. qRT-PCR analysis determined that are repressed ten-fold by the presence thiamine. In total, the data were consistent with a requirement for PLP in thiamine synthesis, perhaps in the Thi5p enzyme, that could only be satisfied by or Additional data showed that Snz3p is a pyridoxal 5'-phosphate synthase and is sufficient to satisfy the pyridoxal 5'-phosphate requirement in when the medium has excess ammonia.
Topics: Pyridoxal Phosphate; Saccharomyces cerevisiae; Thiamine
PubMed: 30498136
DOI: 10.1534/g3.118.200831 -
Biochemistry. Biokhimiia Oct 2002Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an... (Review)
Review
Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme--substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme--substrate compounds and corresponding apoenzyme complexes are reviewed.
Topics: Antibodies, Anti-Idiotypic; Antibodies, Catalytic; Antigens; Apoenzymes; Protein Conformation; Protein Engineering; Pyridoxal Phosphate
PubMed: 12460106
DOI: 10.1023/a:1020955005503 -
Applied and Environmental Microbiology Jun 2019YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family...
YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in -deficient was caused by the pyridoxine 5'-phosphate (PNP)-dependent overproduction of Val, which is toxic to The data suggest that the mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a mutant of Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that mutations in result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the mutant in are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.
Topics: Amino Acids; Biosynthetic Pathways; Carrier Proteins; Escherichia coli; Escherichia coli Proteins; Gene Knockout Techniques; Metabolic Networks and Pathways; Mutation; Pyridoxal Phosphate; Pyridoxine; Transcriptome; Vitamin B 6
PubMed: 30902856
DOI: 10.1128/AEM.00430-19 -
Life Sciences 1988Nonenzymatic glycosylation of serum albumin was studied in the presence of naturally occurring metabolites, pyridoxal, pyridoxal phosphate and...
Nonenzymatic glycosylation of serum albumin was studied in the presence of naturally occurring metabolites, pyridoxal, pyridoxal phosphate and ascorbate/dehydroascorbate, and a hydrazine compound, aminoguanidine. Pyridoxal, pyridoxal phosphate, ascorbate and dehydroascorbate, at concentrations of 0.1 mM or greater, significantly inhibited the nonenzymatic glycosylation of albumin. Aminoguanidine was the most potent inhibitor of nonenzymatic glycosylation and 54% or 85% inhibition occurred when 5 or 50 mM aminoguanidine, respectively, was present in the incubation mixture containing 20 mM glucose. A major effect of aminoguanidine was to lower the free glucose concentration in the incubation mixture by a direct reaction with glucose as judged by thin layer chromatography. The present studies suggest that vital metabolites such as pyridoxal phosphate and ascorbate may be potentially important in controlling glucose-induced nonenzymatic glycosylation of proteins. Pyridoxal phosphate forms a Schiff base with proteins as does glucose and therefore may be a preferable drug, over aminoguanidine which is a hydrazine, for inhibiting the effects of glucose-induced nonenzymatic glycosylation.
Topics: Ascorbic Acid; Glycosylation; Guanidines; Kinetics; Pyridoxal Phosphate; Serum Albumin, Bovine
PubMed: 3193856
DOI: 10.1016/0024-3205(88)90484-5 -
Biochimica Et Biophysica Acta.... Mar 2020Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the...
Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo zebrafish develop seizures resulting in only 38% of pnpo zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.
Topics: Amino Acids; Animals; Brain Diseases, Metabolic; Hypoxia-Ischemia, Brain; Metabolic Diseases; Oxidoreductases; Pyridoxal Phosphate; Pyridoxamine; Pyridoxaminephosphate Oxidase; Seizures; Synaptic Transmission; Zebrafish
PubMed: 31759955
DOI: 10.1016/j.bbadis.2019.165607 -
Current Opinion in Chemical Biology Oct 2009Studies of nonenzymatic electrophilic catalysis of carbon deprotonation of glycine show that pyridoxal 5'-phosphate (PLP) strongly enhances the carbon acidity of... (Review)
Review
Studies of nonenzymatic electrophilic catalysis of carbon deprotonation of glycine show that pyridoxal 5'-phosphate (PLP) strongly enhances the carbon acidity of alpha-amino acids, but that this is not the overriding mechanistic imperative for cofactor catalysis. Although the fully protonated PLP-glycine iminium ion adduct exhibits an extraordinary low alpha-imino carbon acidity (pK(a)=6), the more weakly acidic zwitterionic iminium ion adduct (pK(a)=17) is selected for use in enzymatic reactions. The similar alpha-imino carbon acidities of the iminium ion adducts of glycine with 5'-deoxypyridoxal and with phenylglyoxylate show that the cofactor pyridine nitrogen plays a relatively minor role in carbanion stabilization. The 5'-phosphodianion group of PLP likely plays an important role in catalysis by providing up to 12 kcal/mol of binding energy that may be utilized for transition state stabilization.
Topics: Animals; Biocatalysis; Humans; Molecular Structure; Pyridoxal Phosphate; Thermodynamics
PubMed: 19640775
DOI: 10.1016/j.cbpa.2009.06.023 -
International Journal of Molecular... Jan 2023Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most... (Comparative Study)
Comparative Study
A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl-Induced Mitochondrial Permeability Transition Pore.
Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3U (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.
Topics: Animals; Rats; Calcium; Indicators and Reagents; Lysine; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Permeability; Pyridoxal Phosphate; Rats, Wistar; Sulfhydryl Reagents; Thallium
PubMed: 36768782
DOI: 10.3390/ijms24032460 -
Biochimie Jul 2016The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the... (Review)
Review
Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.
The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.
Topics: Animals; Clinical Trials as Topic; Female; Hep G2 Cells; Humans; Hydrogen Sulfide; Male; Nutritional Status; Pyridoxal Phosphate; Vitamin B 6
PubMed: 26765812
DOI: 10.1016/j.biochi.2015.12.020 -
Applied and Environmental Microbiology Jan 2021Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B, essential for cellular function in all domains of life. In many organisms, such as serovar...
Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B, essential for cellular function in all domains of life. In many organisms, such as serovar Typhimurium and , this cofactor can be synthesized or salvaged from B vitamers in the environment. Unexpectedly, strains blocked in PLP biosynthesis were able to use exogenous PLP and pyridoxine 5'-phosphate (PNP) as the source of this required cofactor, while strains of the same genotype could not. Transposon mutagenesis found that was essential for the salvage of PLP and PNP under the conditions tested. encodes a class A nonspecific acid phosphatase (EC 3.1.3.2) that is transcriptionally regulated by the PhoPQ two-component system. The periplasmic location of PhoN was essential for PLP and PNP salvage, and assays confirmed PhoN has phosphatase activity with PLP and PNP as substrates. The data suggest that PhoN dephosphorylates B vitamers, after which they enter the cytoplasm and are phosphorylated by kinases of the canonical PLP salvage pathway. The connection of with PhoPQ and the broad specificity of the gene product suggest is exploiting a moonlighting activity of PhoN for PLP salvage. Nutrient salvage is a strategy used by species across domains of life to conserve energy. Many organisms are unable to synthesize all required metabolites and must rely exclusively on salvage. Others supplement synthesis with the ability to salvage. This study identified an unexpected mechanism present in that allows salvage of phosphorylated B vitamers. and data herein determined that the periplasmic phosphatase PhoN can facilitate the salvage of PLP and PNP. We suggest a mechanistic working model of PhoN-dependent utilization of PLP and PNP and discuss the general role of promiscuous phosphatases and kinases in organismal fitness.
Topics: Bacterial Proteins; Escherichia coli; Periplasm; Phosphoric Monoester Hydrolases; Pyridoxal Phosphate; Salmonella enterica
PubMed: 33218995
DOI: 10.1128/AEM.02300-20