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)

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities. 137 27

Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.
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PMID:Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical. 138 4

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.
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PMID:Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds. 178 72

Several hundred million tons of toxic mercurials are dispersed in the biosphere. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase and mercuric ion reductase (MerA). The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases, catalyses the reaction NADPH + Hg(II)----NADP+ + H+ + Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), p1258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn501 and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon. These domains can be proteolytically cleaved off without changing the catalytic efficiency. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.
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PMID:Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607. 206 68

A new pathway for aerobic metabolism of 2-aminobenzoate which proceeds via anthranoyl-CoA has recently been revealed in a Pseudomonas strain KB740. This bacterial strain was found to contain a small 8.1-kbp plasmid pKB740 which appears to harbour the genes encoding for two key enzymes catalyzing the initial reactions of the pathway, 2-aminobenzoate coenzyme A ligase and 2-aminobenzoyl-coenzyme A monooxygenase/reductase. The evidence is as follows: The plasmid content of the culture varied by a factor of ten depending on the growth substrates; it was highest when cells were grown aerobically on 2-aminobenzoate. The plasmid pKB740 could be introduced into Escherichia coli strain JM83 by transformation. Wild-type E. coli and E. coli JM83 are unable to metabolize 2-aminobenzoate whereas the transformed E. coli JM83 cells could grow with this aromatic compound as sole organic substrate and oxidize it completely to CO2. The plasmid recovered from E. coli had the same restriction map as the original plasmid, but was dimerized. The two key enzyme activities were demonstrated in the transformed E. coli in sufficiently high amounts to explain growth. They appear to be regulated on the transcription level by induction; they were formed only during aerobic growth in the presence of 2-aminobenzoate, as in the parent Pseudomonas. The N-terminal amino acid sequence of 2-aminobenzoyl-CoA monooxygenase/reductase was similar to the consensus sequence of the FAD binding site of different flavoenzymes. The data also prove that the enzyme with two flavin functions is a alpha 2 homodimer. Southern blotting of digested chromosomal and plasmid DNA and hybridization against a labelled 15-base oligonucleotide derived from the N-terminal amino acid sequence of 2-aminobenzoyl-CoA monooxygenase/reductase revealed that the gene for this enzyme is coded on the plasmid rather than on the chromosome. The gene was localized on a 3.2-kbp restriction fragment. The formation of 2-aminobenzoyl-CoA monooxygenase/reductase protein in transformed E. coli was demonstrated by Western blotting of proteins of cell extracts separated by SDS/PAGE. The enzyme protein band, which was stained by a procedure based on antibodies against 2-aminobenzoyl-CoA monooxygenase/reductase, was demonstrated in transformed E. coli.
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PMID:Evidence that enzymes of a novel aerobic 2-amino-benzoate metabolism in denitrifying Pseudomonas are coded on a small plasmid. 217 2

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.
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PMID:Quaternary structure and composition of squash NADH:nitrate reductase. 403 8

Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.
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PMID:Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. 645 37

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subunit dissociation and unfolding of macrophage NO synthase: relationship between enzyme structure, prosthetic group binding, and catalytic function. 754 34

Inducible macrophage NO synthase (iNOS) is a homodimer of 130 kDa subunits. Trypsinolysis of iNOS inactivates its NO synthesis activity and cleaves the enzyme into a dimeric oxygenase fragment that contains heme, tetrahydrobiopterin, and the substrate binding site and a monomeric reductase fragment that contains FAD, FMN, calmodulin, and the binding site for NADPH [Ghosh, D. I., & Stuehr, D. H. (1995) Biochemistry 34, 801-807]. In this paper, we describe the reconstitution of NO synthesis activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not produced from L-arginine but could be formed from the reaction intermediate N omega-hydroxy-L-arginine (L-NOHA). The apparent Km with L-NOHA in the reconstituted system was 100 microM versus 19 microM for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occurred at an oxygenase to reductase molar ratio of 4:1, with higher ratios causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an incomplete reduction of the oxygenase domain heme iron. L-NOHA, but not L-arginine, increased NADPH consumption in the reconstituted system. Between 2.5 and 3.0 NADPH were consumed per NO formed from L-NOHA, considerably higher than the stoichiometry obtained with native iNOS (0.5 NADPH oxidized per NO formed), indicating an uncoupled electron transfer between the domain fragments. Thus, the isolated iNOS reductase and oxygenase domains each retain their separate catalytic functions but interact to catalyze only the second step of NO synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reconstitution of the second step in NO synthesis using the isolated oxygenase and reductase domains of macrophage NO synthase. 754 58

2-Aminobenzoyl-CoA monooxygenase/reductase catalyzes both monooxygenation and hydrogenation of anthraniloyl-CoA. Its reactivity with 11 substrate analogs has been investigated. Only 2-aminobenzoyl-CoA (anthraniloyl-CoA) in its normal and deuterated (5-2H) form is a full substrate, and only traces of 2-hydroxybenzoyl-CoA (salicyloyl-CoA) are probably monooxygenated but not hydrogenated. The purified enzyme is a homodimer and has been resolved preparatively into three major species by anion-exchange chromatography on Mono Q. All three species have the same specific activity when reconstituted to full content of FAD, they differ, however, substantially in their mode of binding FAD. The oxidized or fully reduced enzyme forms bind tightly 0.5 mol/mol of the substrate 2-aminobenzoyl-CoA (Kd = 1-2 microM). The enzyme can be depleted of approximately 50% of its FAD, which corresponds to essentially complete removal from one of the two binding sites, reflecting a large difference in the affinity for FAD. From this it is deduced that the two sites are not equivalent. Removal of FAD from one binding site leads to loss of the hydrogenation capacity of the enzyme, while monooxygenation catalysis is retained. The FAD cofactors of the two binding sites differ drastically in their reactivities towards NADH, oxygen and N-ethylmaleimide. Exchange of reducing equivalents between the two FAD cofactors at the respective binding sites is very slow and irrelevant compared to the rates of catalysis. It is concluded that the enzyme, which has been proposed to consist of two identical polypeptide chains [Altenschmidt, U., Bokranz, M. & Fuchs, G. (1992) Eur. J. Biochem. 207, 715-722], contains two active centers which differ substantially in their catalytic activity. One center belongs to the class of monooxygenases, the other one to the (de)hydrogenases. This must result from substantially different interaction of the same flavin cofactors with protein functional groups and is, to our knowledge, unprecedented in flavoprotein enzymology.
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PMID:2-Aminobenzoyl-CoA monooxygenase/reductase. Evidence for two distinct loci catalyzing substrate monooxygenation and hydrogenation. 760 42


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