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)

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.
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PMID:Identification of the domains of neuronal nitric oxide synthase by limited proteolysis. 866 Mar 10

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.
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PMID:Characterization of C415 mutants of neuronal nitric oxide synthase. 867 77

Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.
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PMID:Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself. 870 5

Nitric oxide (NO) was discovered to be a potent vasodilator, inhibitor of platelet aggregation, and active species of nitroglycerin before the discovery of endothelium-derived relaxing factor (EDRF) in 1980. Subsequent studies revealed that EDRF is NO, and is synthesized by mammalian cells from L-arginine through a complex oxidation reaction catalyzed by the flavo-hemoprotein NO synthase (NOS). NOS catalyzes the NADPH- and oxygen-dependent oxygenation of L-arginine to NO plus L-citrulline in a reaction that requires at least six cofactors including NADPH, FAD, FMN, tetrahydrobiopterin, heme, and calmodulin. NO elicits its known physiological actions by activating cytosolic guanylate cyclase, which converts GTP to cyclic GMP. Endothelial NOS and neuronal NOS are constitutively present and activated by increases in intracellular calcium triggered by endogenous chemicals. NO then diffuses into nearby target cells to elevate cyclic GMP levels and thereby trigger cell function. NOS activity can also be regulated by a negative feedback mechanism involving NO itself. Much greater quantities of NO are produced pathophysiologically by a distinct form of NOS that can be induced in vascular endothelium, smooth muscle and macrophages by endotoxin and cytokines. This high-output production of NO is not regulated by calcium and is cytotoxic by mechanisms involving interaction with iron-containing proteins.
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PMID:Physiology and pathophysiology of nitric oxide. 874 1

Nitric oxide, derived from L-arginine by the enzyme nitric oxide synthase, is an activator of the soluble guanylate cyclase and a cellular messenger. This work demonstrates that, in cat brain, the neuronal constitutive nitric oxide synthase activity is a) NADPH/calcium dependent, b) independent upon exogenous calmodulin in crude brain supernatant, c) significantly enhanced by exogenous FAD and tetrahydrobiopterin (Vmax: 118 instead of 59.4 pmol of citrulline formed .mg of prot.-1 min-1, d) inhibited by calcium chelators and calmodulin antagonist, and e) present in several neuroanatomical structures. Moreover, the Km value for L-arginine was of 11 microM instead of 41 microM in the presence of FAD and tetrahydrobiopterin in the incubation mixture, thus demonstrating that these cofactors are able to stabilize the enzyme-substrate interactions.
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PMID:Nitric oxide synthase in cat brain: cofactors--enzyme-substrate interaction. 879 Oct 99

The increase in cyclic GMP contents in phototransduction is likely to be mediated with release of nitric oxide (NO). In this study we have characterized a constitutive NO synthase (NOS) isolated from rat retina supernatant. The activity of NOS was determined by monitoring L-citrulline formation from L-arginine. Soluble NOS from retina used L-arginine as substrate, with NADPH, tetrahydrobiopterin and FAD as cofactors. The enzyme activity was raised with additional calcium and was reduced with high concentrations of calmodulin inhibitors. Protein immunoblot and immunohistochemical analysis using a specific antibody against Type I NOS indicated that the same type of NOS is mainly localized in amacrine cells in the retina. However, the enzyme activity in freshly prepared sample was not completely abolished in the absence of calcium or with calmodulin inhibitors. In addition, NADPH diaphorase staining in retina was wider and more intensive than staining with the antibody against Type I NOS. These results indicate that rat retina contains more than one type of NOS.
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PMID:Biochemical and immunohistochemical characterization of nitric oxide synthase in the rat retina. 881 43

Endothelial nitric-oxide synthase (eNOS) is comprised of two identical subunits. Each subunit has a bidomain structure consisting of an N-terminal oxygenase domain containing heme and tetrahydrobiopterin (BH4) and a C-terminal reductase domain containing binding sites for FAD, FMN, and NADPH. Each subunit is also myristoylated and contains a calmodulin (CaM)-binding site located between the oxygenase and reductase domains. In this study, wild-type and mutant forms of eNOS have been expressed in a baculovirus system, and the quaternary structure of the purified enzymes has been analyzed by low temperature SDS-PAGE. eNOS dimer formation requires incorporation of the heme prosthetic group but does not require myristoylation or CaM or BH4 binding. In order to identify domains of eNOS involved in subunit interactions, we have also expressed eNOS oxygenase and reductase domain fusion proteins in a yeast two-hybrid system. Corresponding human neuronal NOS (nNOS) and murine inducible NOS (iNOS) fusion proteins have also been expressed. Comparative analysis of NOS domain interactions shows that subunit association of eNOS and nNOS involves not only head to head interactions of oxygenase domains but also tail to tail interactions of reductase domains and head to tail interactions between oxygenase and reductase domains. In contrast, iNOS subunit association involves only oxygenase domain interactions.
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PMID:Subunit interactions of endothelial nitric-oxide synthase. Comparisons to the neuronal and inducible nitric-oxide synthase isoforms. 899 32

Rhodnius prolixus, a blood-sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132,331 Da. The primary structures of mammalian NOS, including the putative cofactor-recognition sites for heme, tetrahydrobiopterin (BH4), calmodulin. FMN, FAD and NADPH are all conserved in salivary-gland NOS. Recombinant salivary-gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N-terminal domain and an N-terminal myristylation sequence, respectively. Salivary-gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary-gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary-gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.
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PMID:cDNA cloning, expression and characterization of nitric-oxide synthase from the salivary glands of the blood-sucking insect Rhodnius prolixus. 902 13

The active form of endothelial nitric-oxide synthase (eNOS) is a homodimer. The activity of the enzyme is regulated in vivo by calcium signaling involving the binding of calmodulin (CAM), which triggers the activation of eNOS. We have examined the possible role of calcium-mediated CAM binding in promoting dimerization of eNOS through the oxygenase domain of the enzyme. A recombinant form of the oxygenase domain of human eNOS was expressed in a prokaryotic expression system. This recombinant domain contains the catalytic cytochrome P-450 site for arginine oxidation by molecular oxygen as well as the binding sites for tetrahydrobiopterin and Ca2+-CAM but lacks the reductase domain and associated FAD, FMN, and NADPH binding sites. Binding of Ca2+-CAM caused an association of monomeric eNOS oxygenase domain as determined by changes in fluorescence, both intrinsic and extrinsic, and by gel filtration, chemical cross-linking, and particle-sizing. Dimerization of the domain was not dependent on the presence of the substrate, arginine, or the cofactor, tetrahydrobiopterin. A truncated form of the eNOS oxygenase domain lacking the Ca2+-CAM binding region did not undergo self-association to form dimers. These results show that the eNOS reductase domain is not required for Ca2+-CAM-induced dimerization of eNOS and suggest that this dimerization may be a primary event in the activation of eNOS by Ca2+.
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PMID:Calmodulin promotes dimerization of the oxygenase domain of human endothelial nitric-oxide synthase. 911 69

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. By using domain boundaries defined by limited proteolysis of full-length enzyme, recombinant haem-binding regions of rat brain neuronal nitric oxide synthase were expressed and purified. Two proteins were made in high yield: one, corresponding to residues 221-724, contained bound haem and tetrahydrobiopterin and was able to bind Nomega-nitro-l-arginine (nitroarginine) or arginine; the other, containing residues 350-724, contained bound haem but was unable to bind tetrahydrobiopterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for arginine/tetrahydrobiopterin binding between residues 221 and 350. Limited proteolysis with chymotrypsin of the former protein resulted in a new species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and cofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site per dimer.
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PMID:Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase. 917 72


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