Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.1 (NADPH-diaphorase)
 3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.
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PMID:Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase. 281 48

The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
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PMID:Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. 836 77

Scanning electron microscopic (SEM) observation demonstrates the differentiation of mesocarp and endocarp tissues and their lignified nature in dura fruits at 8 weeks after pollination (WAP). During shell formation, the endocarp cells become lignified to a hard shell while the mesocarp tissue remains cellular and fibrous. A transition zone made up of fibrous units was also visible beneath the shell. The soluble phenols of mesocarp and endocarp tissues at their developmental stage was analyzed using Reverse phase high performance liquid chromatography (RP-HPLC). The appearance of ferulic acid at 4 WAP and its absence at 8 WAP indicates the role of ferulic acid in lignin synthesis. The HPLC data was supported by the lignin concentration. To ascertain the biochemical relationship of lignin pathway enzymes, phenylalanine ammonia lyase (PAL), cinnamyl alcohol-NADPH-dehydrogenase (CAD) and peroxidase (POD) with shell synthesis, the activities of these enzymes and lignin content were assessed during development of the shell between 4 and 8 WAP. The three enzymes, PAL, CAD and POD expressed high level of activity in the mesocarp and endocarp at 4 WAP. At 8 WAP a sharp decline in activity was observed in the endocarp whereas the mesocarp showed a moderate reduction. This variation is an indication of the role of these enzymes in shell formation.
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PMID:Association of lignifying enzymes in shell synthesis of oil palm fruit (Elaeis guineensis--dura variety). 1148 Feb 13

The retinopetal neurons of Crocodylus niloticus were visualized by retrograde transport of rhodamine beta-isothiocyanate or Fast Blue administered by intraocular injection. Approximately 6,000 in number, these neurons are distributed in seven regions extending from the mesencephalic tegmentum to the rostral rhombencephalon, approximately 70% being located contralaterally to the injected eye. None of the centrifugal neurons projects to both retinae. The retinopetal neurons are located in rostrocaudal sequence in seven regions: the formatio reticularis lateralis mesencephali, the substantia nigra, the griseum centralis tectalis, the nucleus subcoeruleus dorsalis, the nucleus isthmi parvocellularis, the locus coeruleus, and the commissura nervi trochlearis. The greatest number of cells (approximately 93%) is found in the nucleus subcoeruleus dorsalis. The majority are multipolar or bipolar in shape and resemble the ectopic centrifugal visual neurons of birds, although a small number of monopolar neurons resembling those of the avian isthmo-optic nucleus may also be observed. A few retinopetal neurons in the griseum centralis tectalis were tyrosine hydroxylase (TH) immunoreactive. Moreover, in the nuclei subcoeruleus dorsalis and isthmi parvocellularis, both ipsilaterally and contralaterally, approximately one retinopetal neuron in three (35%) was immunoreactive to nitric oxide synthase (NOS), and a slightly higher proportion (38%) of retinopetal neurons were immunoreactive for choline acetyltransferase (ChAT). Some of them contained colocalized ChAT and NOS/reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Fibers immunoreactive to TH, serotonin (5-HT), neuropeptide Y (NPY), or Phe-Met-Arg-Phe-amide (FMRF-amide) were frequently observed to make intimate contact with rhodamine-labeled retinopetal neurons. These findings are discussed in relation to previous results obtained in other reptilian species and in birds.
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PMID:Centrifugal visual system of Crocodylus niloticus: a hodological, histochemical, and immunocytochemical study. 1464 91

Plastid gene expression is regulated by a variety of nuclear genes. We have isolated Arabidopsis thaliana proton gradient regulation 3 (pgr3) mutants, which display aberrant chlorophyll fluorescence because of defects in chloroplast gene expression. High chlorophyll fluorescence (HCF) because of a reduced level of the cytochrome b6/f complex was observed in two alleles, pgr3-1 and pgr3-2 but not in pgr3-3. In contrast, a transient increase in fluorescence after turning off the actinic light, which was ascribed to chloroplast NADPH dehydrogenase (NDH) activity, was impaired in pgr3-1 and pgr3-3 but not in pgr3-2. Both phenotypes were complemented by the introduction of a single gene, PGR3, encoding a protein containing 27 pentatrico-peptide repeat (PPR) motifs. PPR motifs are present in proteins functioning in the post-transcriptional regulation of organellar gene expression. The conserved threonine in the motif was substituted by isoleucine in the 15th and 12th PPR motifs in pgr3-1 and pgr3-2, respectively, and the conserved leucine by phenylalanine in the final incomplete motif of pgr3-3. We consider that the different domains of the PPR repeats in PGR3 might have different functions in conferring RNA stability and probably allowing translation as well as recognizing at least two distinct target RNAs.
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PMID:PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis. 1505 68

The possible involvement of the L-arginine-containing Phe-met-arg-phe (FMRF)-amide (FMRFa) in neuronal nitric oxide (NO) biosynthesis was studied in a gastropod species. We found NADPH-diaphorase-positive neurons and FMRFa-containing fibers in close proximity in the enteric nervous system. Administration of L-arginine and FMRFa induced quantitatively similar nitrite production in both intact intestinal tissues and tissue homogenates. These changes could be prevented by the presence of NOARG (an NO synthase inhibitor). Neither chemically modified FMRFa (D-arginine instead of L-arginine) nor amino acid constituents of FMRFa (methionine, phenylalanine) affected basal nitrite production. FMRFa-induced alterations were reduced in the presence of Na+ channel blockers (tetrodotoxin, amiloride, lidocaine), the Na+/K+ATPase inhibitor ouabain, or protease inhibitors (leupeptine, pepstatine-a). FMRFa and its amino acid constituents were analyzed by paper chromatography. When FMRFa was added to tissue homogenates, the peptide was eliminated within 1-2 min, whereas methionine, phenylalanine, arginine, and citrulline levels were elevated simultaneously. We tested the effects of FMRFa, L-arginine, and NOARG on intestinal contractile activity. FMRFa relaxed the intestine for 1-2 min and then induced contractions for 20-40 min. In the presence of NOARG, no relaxant effect of FMRFa was recorded. As administration of L-arginine strongly inhibits the mechanical activity of the intestinal muscle, NO production presumably plays a substantial role in the action of FMRFa, at least in the initial phase. Our biochemical data indicate a direct involvement of FMRFa in NO biosynthesis. FMRFa might be hydrolyzed by extracellular peptidases and then the locally released arginine might be transported into the cells and broken-down to produce NO. Depolarization-induced NO production attributable to the activation of amiloride-sensitive Na+ channels might also be involved.
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PMID:Phe-met-arg-phe (FMRF)-amide is a substrate source of NO synthase in the gastropod nervous system. 1661 29

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.
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PMID:Application of an enzyme chip to the microquantification of l-phenylalanine. 1704 6

In order to clarify the poorly understood mechanisms of two-electron reduction of quinones by flavoenzymes, we examined the quinone reductase reactions of a member of a structurally distinct old yellow enzyme family, Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase (PETNR). PETNR catalyzes two-electron reduction of quinones according to a 'ping-pong' scheme. A multiparameter analysis shows that the reactivity of quinones increases with an increase in their single-electron reduction potential and pK(a) of their semiquinones (a three-step (e(-),H(+),e(-)) hydride transfer scheme), or with an increase in their hydride-transfer potential (E(7)(H(-))) (a single-step (H(-)) hydride transfer scheme), and decreases with a decrease in their van der Waals volume. However, the pH-dependence of PETNR reactivity is more consistent with a single-step hydride transfer. A comparison of X-ray data of PETNR, mammalian NAD(P)H : quinone oxidoreductase (NQO1), and Enterobacter cloacae nitroreductase, which reduce quinones in a two-electron way, and their reactivity revealed that PETNR is much less reactive, and much less sensitive to the quinone substrate steric effects than NQO1. This may be attributed to the lack of pi-pi stacking between quinone and the displaced aromatic amino acid in the active center, e.g., with Phe-178' in NQO1.
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PMID:Two-electron reduction of quinones by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships. 1754 2

With a view to their use in the kinetic resolution of racemic non-natural amino acids, five variants of the enzyme L-phenylalanine dehydrogenase, the wild-type enzyme from Bacillus sphaericus and four active-site mutants, have been tested with a range of amino acids. In each case, the rates of reaction with 0.2 mM L-amino acid and with the racemic mixture at 0.4 mM were compared, so that the starting concentration of the active substrate was kept constant. Although the D-amino acids are not substrates, they were inhibitory in all cases. The extent of inhibition, however, varied greatly from compound to compound and among the mutants. With the N145L mutant and DL 4-O-Me-Phe, the equimolar D-enantiomer gave 83.2% inhibition, and with the wild-type enzyme there was 86.7% inhibition with racemic norleucine. By contrast, with these same substrates the N145V mutant showed less than 9% and 24% inhibition respectively. The N145A mutant was selected for use with DL-4-Cl-Phe. The pH was decreased from the enzyme's optimum of 10.4 to 9.5 to minimise breakdown of the coenzyme NAD(+), and the coenzyme was recycled by molecular oxygen with the assistance of a commercial diaphorase. Reaction on a 200 micromole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 microg N145A enzyme and 100 microg diaphorase, was monitored by chiral HPLC. The L-isomer was removed to an extent of >99% after 40 h, with the D-isomer peak undiminished. The pure D-isomer was isolated from the reaction mixture in 85% overall yield after ion-exchange chromatography.
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PMID:Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the D-enantiomer from racemic mixtures. 1908 64

Site-saturation mutagenesis was used to generate all possible replacements for Trp 116 of Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis ) old yellow enzyme (OYE). Our original hypothesisthat smaller amino acids at position 116 would allow better acceptance of bulky 3-alkyl-substituted 2-cyclohexenonesproved incorrect. Instead, Phe and Ile replacements favored the binding of some substrates in an opposite orientation, which yielded reversed stereochemical outcomes compared to that of the wild-type OYE. For example, W116I OYE reduced (R)- and (S)-carvone to enantiomeric products, rather than the diastereomers produced by the wild-type OYE. Deuterium labeling revealed that (S)-carvone reduction by the W116I OYE occurred by the same pathway as that by the wild type (net trans-addition of H(2)), proving that different substrate binding orientations were responsible for the divergent products. Trp 116 mutants also afforded different stereochemical outcomes for reductions of (R)-perillaldehyde and neral. Preliminary studies of an OYE family member whose native sequence contains Ile at position 116 ( Pichia stipitis OYE 2.6) revealed that this enzyme's stereoselectivity matched that of the wild-type S. pastorianus OYE, showing that the identity of the residue at position 116 does not solely determine the substrate binding orientation. Computational docking studies using an induced fit methodology successfully reproduced the majority of the experimental outcomes. These computational tools will allow preliminary in silico screening of additional residues to identify those most likely to control the substrate binding orientation and provide some guidance to future experimental studies.
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PMID:Site-saturation mutagenesis of tryptophan 116 of Saccharomyces pastorianus old yellow enzyme uncovers stereocomplementary variants. 1922 27


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