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At least 289 records · Page 16Linked to original sources
PubMed2000

Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein.

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.

Animals↗
PubMed2026

Pyridoxal-phosphate-dependent biosynthesis of aminovaleramide by AvaS in tRNA.

In eubacteria, decoding of isoleucine codon AUA requires a specialized tRNA (tRNAIle2) modified with lysidine (k2C) at the anticodon wobble position (C34), which switches decoding specificity from methionine (AUG) to isoleucine (AUA). Recently, aminovaleramide cytidine (ava2C) was discovered at the same tRNA position in several bacteria and plants and shown to support AUA decoding and Ile-specific aminoacylation. However, the enzyme catalyzing ava2C was unknown. Here, we report that tRNAIle-aminovaleramididine synthetase (AvaS) catalyzes ava2C biosynthesis in Pseudomonas aeruginosa PA14. AvaS converts k2C to ava2C through a pyridoxal-phosphate-dependent oxidative decarboxylation mechanism, supported by site-directed mutagenesis and in vitro enzymatic assays. Dual-reporter assays demonstrated that ava2C-modified tRNA exhibits lower AUA decoding efficiency than k2C-modified tRNA. Additionally, genome-wide screening revealed an unexpected link between ava2C levels and metabolic and stress response pathways influencing i6A/ms2i6A dynamics. Together, these findings define the molecular basis of ava2C biosynthesis and its broader cellular metabolic networks.

Journal Article↗
PubMed1991

Pyridoxal-5'-phosphate and pyridoxal biokinetics in aging Wistar rats.

Biokinetic parameters of plasma pyridoxal-5'-phosphate (PLP) and pyridoxal (PL) disposition were studied in male Wistar rats aged 8 and 27 months kept from weaning on a purified diet containing 250 g casein and 6 mg pyridoxine.HCl per kg. Baseline plasma PLP concentration was lower in the older animals (514 +/- 56 nmol/L for young and 317 +/- 124 nmol/L for old animals), whereas baseline plasma PL concentration did not differ between age groups (average 235 nmol/L for both young and old animals). We hypothesized lower baseline plasma PLP in the older animals was caused by an increased PLP elimination rate, a decreased PLP synthesis rate, or a combination of these processes. Observations from earlier in vitro experiments suggest age-related changes occur in vitamin B-6 metabolizing enzyme activities. In the in vivo experiments described here no age-related difference in plasma PLP elimination rate nor in plasma PLP synthesis rate was observed to explain the observed decrease in plasma PLP concentration with age.

Aging↗
PubMed1991

Pyridoxal-5'-phosphate and pyridoxal biokinetics in male Wistar rats fed graded levels of vitamin B-6.

Biokinetic parameters of plasma pyridoxal-5'-phosphate (PLP) and pyridoxal (PL) disposition were studied in male Wistar rats (age 8 mo) fed a purified diet containing less than 0.5, approximately 3 or approximately 6 mg pyridoxine.HCl/kg diet from weaning, with animals fed the 6 mg/kg diet serving as the control group. Basal plasma PLP concentration was lower in both the less than 0.5 and 3 mg/kg diet groups than in control animals (98 +/- 12, 314 +/- 40 and 514 +/- 56 nmol/L, respectively). Basal plasma PL concentration was lower in the less than 0.5 mg/kg diet group only [60 nmol/L (measured in pooled samples), 190 +/- 73 and 235 +/- 63 nmol/L for less than 0.5, 3 and 6 mg/kg diet groups, respectively]. In both the less than 0.5 and 3 mg/kg diet groups, PLP clearance was lower than in control rats (0.158 +/- 0.025, 0.131 +/- 0.040 and 0.240 +/- 0.051 L.h-1.kg body weight-1, respectively). In the less than 0.5 mg/kg diet group, PLP synthesis was more efficient than in control animals (34.7 +/- 9.3, 12.1 +/- 2.5 and 16.7 +/- 11.4% for less than 0.5, 3 and 6 mg/kg diet groups, respectively). In both the less than 0.5 and 3 mg/kg diet groups, volume of distribution of PLP as well as of PL was larger than in controls. It is concluded that B-6 vitamer metabolism is influenced by vitamin B-6 status. The metabolic pathway involved (PLP synthesis and/or PLP degradation) was observed to depend on degree of vitamin B-6 deficiency.

Animals↗
PubMed2001

Pyridoxal 5'-phosphate and pyridoxal inhibit angiogenesis in serum-free rat aortic ring assay.

Supraphysiological doses of vitamin B6 has been reported to suppress tumor growth and metastasis in rodents. To examine if its anticancer effect is due to suppression in angiogenesis, this study was conducted to investigate the antiangiogenic effect of pyridoxal 5'-phosphate (PLP), pyridoxine, pyridoxal and pyridoxamine in an ex vivo serum-free matrix culture model using rat aortic ring. Rat aortic rings were incubated with PLP or pyridoxine (25 micromol/l to 5 mmol/l). Higher concentrations of PLP (2.5 and 5 mmol/l) and pyridoxine (5 mmol/l) caused complete inhibition of microvessel outgrowth. However, the addition of pyridoxine at 2.5 mmol/l did not show complete inhibition of angiogenesis. PLP inhibited microvessel outgrowth almost completely at a concentration of 500 micromol/l and showed antiangiogenic effect in a dose-dependent manner within the range of 25-500 micromol/l. At 250 micromol/l, pyridoxal was as effective as PLP, but pyridoxamine was inactive, implying that the aldehyde group relates to the antiangiogenic effect. These results indicated the antiangiogenic effect of PLP and pyridoxal, and suggested that the antitumor effect of high levels of vitamin B6 might be mediated through suppression of angiogenesis.

Angiogenesis Inhibitors↗
PubMed1989

Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine.

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.

Adenosine Diphosphate↗
PubMed1990

2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme.

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.

Acetyltransferases↗
PubMed1980

Externally disposed polypeptides of chick synaptosomal plasma membrane. Identification with pyridoxal phosphate and sodium borotritide.

The topographical distribution of polypeptides in chick brain synaptic plasma membrane was studied using pyridoxal phosphate-sodium borotritide labeling. Labeling of intact synaptosomes was restricted to the external surface only by very careful adjustment of the reaction conditions. Fourteen major external polypeptides were labeled. These had apparent molecular weights of 210 000, 160 000, 130 000, 100 000, 92 000, 82 000, 60 000, 52 000, 42 000, 34 000, 29 000, 26 000, 24 000, and 19 000. Most of the label was incorporated into the 42 000, 29 000 and 26 000 dalton polypeptides.

Animals↗
PubMed1984

Characterization of the estrogen receptor extracted from hen oviduct nuclei with pyridoxal phosphate.

The estrogen receptor was extracted in high yield from nuclei of laying hen oviduct with 10 mM pyridoxal-5'-phosphate (PLP). The receptor extracted under these conditions, unlike that extracted with 0.4 M KCl, displayed no tendency to aggregate on sucrose gradients in low salt. The receptor was eluted as a single peak from DEAE-Sephacel at an ionic strength of 0.13 M KCl. The receptor after DEAE chromatography had approximately half the molecular weight of that in the nuclear extract. A larger form could be reconstituted by the addition of whole nuclear extract to the DEAE eluate. These data support the notion that the nuclear estrogen receptor is a dimer composed of similar subunits.

Animals↗
PubMed2001

Production of pyridoxal phosphate by a mutant strain of Schizosaccharomyces pombe.

Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.

Air↗
PubMed1990

Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.

In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild-type enzyme but at attenuated rates, may yield information on the factors controlling the stereochemistry of the reaction as well as on the catalytic steps of the transaminase pathway.

Base Sequence↗
PubMed1987

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors.

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗