Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxalate oxidase (OXO) was purified to homogeneity in three steps from roots of barley seedlings. The purification method comprised: (i) thermal treatment (60 degrees C, 10 min), (ii) affinity chromatography on immobilized either Procion turquoise MX-G dye or biomimetic aminoethyl oxamic blue dye, and (iii) affinity chromatography on immobilized lectin concanavalin A (overall performance: 1096-fold purification, 42% recovery). The purified enzyme has a specific activity of 34 U mg-1 (25 degrees C), and is a homopentamer of M(r) approximately 125,000 (HPLC analysis) showing a single band on SDS-polyacryl-amide gel electrophoresis (M(r) approximately 26,000) after staining with silver nitrate. The kinetic constants of the purified enzyme for oxalate are K(m) 0.27 mM and kcat 22 s-1 (37 degrees C), whereas at [oxalate] > or = 4 mM the enzyme exhibited substrate inhibition. Barley root OXO contains no prosthetic group absorbing at 370 or 450 nm, and riboflavin and FAD have no effect on its activity. The enzyme is activated by 1 mM each of Ca2+ (1.7-fold) and Pb2+ (2.6-fold). Irreversible inactivation studies with denatured (70 degrees C) and native (37 degrees C) enzyme using the sulfhydryl-attacking reagent 5,5-dithiobis(2-nitrobenzoic) acid (1.4 mM), in the presence and absence of SDS, respectively, have shown that denatured OXO (4% SDS, 10 min, 100 degrees C) exhibited 10 HS groups per molecule, whereas native OXO displayed one accessible HS group per molecule after approximately 15 min incubation and, over the same period, maintained its catalytic activity to 90%. Furthermore, native OXO treated with beta-mercaptoethanol (1 mM) lost 83% of its catalytic activity within 5 min. These findings indicate that some cysteines may preserve the catalytic activity of OXO by maintaining the integrity of its tertiary structure via disulfide bond formation.
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PMID:Oxalate oxidase from barley roots: purification to homogeneity and study of some molecular, catalytic, and binding properties. 914 27

The assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.
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PMID:The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein. 930 29

Nitrate is a significant nitrogen source for plants and microorganisms. Recent molecular genetic analyses of representative bacterial species have revealed structural and regulatory genes responsible for the nitrate-assimilation phenotype. Together with results from physiological and biochemical studies, this information has unveiled fundamental aspects of bacterial nitrate assimilation and provides the foundation for further investigations. Well-studied genera are: the cyanobacteria, including the unicellular Synechococcus and the filamentous Anabaena; the gamma-proteobacteria Klebsiella and Azotobacter; and a Gram-positive bacterium, Bacillus. Nitrate uptake in most of these groups seems to involve a periplasmic binding protein-dependent system that presumably is energized by ATP hydrolysis (ATP-binding cassette transporters). However, Bacillus may, like fungi and plants, utilize electrogenic uptake through a representative of the major facilitator superfamily of transport proteins. Nitrate reductase contains both molybdenum cofactor and an iron-sulfur cluster. Electron donors for the enzymes from cyanobacteria and Azotobacter are ferredoxin and flavodoxin, respectively, whereas the Klebsiella and Bacillus enzymes apparently accept electrons from a specific NAD(P)H-reducing subunit. These subunits share sequence similarity with the reductase components of bacterial aromatic ring-hydroxylating dehydrogenases such as toluene dioxygenase. Nitrite reductase contains sirohaem and an iron-sulfur cluster. The enzymes from cyanobacteria and plants use ferredoxin as the electron donor, whereas the larger enzymes from other bacteria and fungi contain FAD and NAD(P)H binding sites. Nevertheless, the two forms of nitrite reductase share recognizable sequence and structural similarity. Synthesis of nitrate assimilation enzymes and uptake systems is controlled by nitrogen limitation in all bacteria examined, but the relevant regulatory proteins exhibit considerable structural and mechanistic diversity in different bacterial groups. A second level of control, pathway-specific induction by nitrate and nitrite in Klebsiella, involves transcription antitermination. Several issues await further experimentation, including the mechanism and energetics of nitrate uptake, the pathway(s) for nitrite uptake, the nature of electron flow during nitrate reduction, and the action of transcriptional regulatory circuits. Fundamental knowledge of nitrate assimilation physiology should also enhance the study of nitrate metabolism in soil, water and other natural environments, a challenging topic of considerable interest and importance.
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PMID:Nitrate assimilation by bacteria. 932 45

This study reports the molecular characterization of the nitrate-assimilation gene cluster from the opportunistic fungal pathogen Aspergillus fumigatus. A genomic fragment was isolated which contained the entire structural gene encoding nitrite reductase (niiA), plus segments of the nitrate reductase (niaD) and the nitrate transporter (crnA) genes. Nitrate-assimilation genes in A. fumigatus are physically linked and transcribed in the same direction as in A. nidulans. The nitrate-assimilation gene cluster is on the largest chromosome (5.3 Mb). The nitrite reductase (niiA) gene encodes a protein of 1110 amino acids that contains regions corresponding to FAD, NADPH, FeS and siroheme binding sites. Eight small introns interrupt the niiA open reading frame. The niaD-niiA intergenic regulatory region contains promoter consensus sequences including TATA, CAAT, and binding sites for the areA and nirA gene products. Northern analysis indicated that the expression of niaD, niiA and crnA are induced by nitrate and repressed by ammonium at the transcriptional level.
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PMID:Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus. 950 95

Hansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit- (nitrate-) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR- mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.
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PMID:Nitrate reduction and the isolation of Nit- mutants in Hansenula polymorpha. 972 55

Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO*. An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3-. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO*. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO*. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO* detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage.
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PMID:Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. 972 11

Nitric oxide (NO) synthesis is well-known to result from the oxidation of L-arginine by a family of NO synthases (NOS). However, under hypoxic conditions this mechanism of NO synthesis may be impaired and NO is formed by a NOS independent mechanism. This study was designed to examine the reduction of nitrite to NO by xanthine oxidase (XO) under hypoxia, because the bacterial nitrate/nitrite reductases have structural similarity to XO. We found that both purified and tissue containing XO catalyze the reduction of nitrite to NO, as demonstrated using a chemiluminescent NO meter. This redox reaction requires NADH as an electron donor, and is oxygen independent. The inhibitory profiles suggest that reduction of nitrite takes place at the molybdenum center of XO whilst NADH is oxidized at the FAD center. Heparin binding of XO caused an increase in the catalysis of nitrite reduction. The XO-catalyzed generation of NO may be important in redistribution of blood flow to ischaemic tissue as a supplement to NOS, since both nitrite and NADH have been shown to be elevated in hypoxic tissue.
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PMID:Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity. 973 Dec 11

Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.
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PMID:Single-cell analyses of nitrergic neurons in simple nervous systems. 991 42

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.
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PMID:The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S]. 1065 2

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V(max)) of 670 s(-1) and K(m) values for NADH, NO, and O(2) equal to 4.8, 0.28, and approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-1) s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 microM(-1) s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O(2), dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O(2)] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O(2) dissociation rate constant approximately 80-fold and reduces NO dioxygenase activity approximately 30-fold, demonstrating the importance of the tyrosine hydroxyl for O(2) affinity and NO scavenging activity. At 37 degrees C, V(max)/K(m)(NO) is 2,400 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.
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PMID:Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis. 1077 48


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