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)

The cytokine-inducible NO synthase (iNOS) is a flavin-containing hemeprotein that must dimerize to generate NO. Trypsin cleaves the dimeric enzyme into an oxygenase domain fragment that remains dimeric, contains heme and H4biopterin, and binds L-arginine and a reductase domain fragment that is monomeric, binds NADPH, FAD, FMN, and catalyzes the reduction of cytochrome c [Ghosh, D. K. & Stuehr, D. J. (1995) Biochemistry 34, 801-807]. The current study investigates the isolated oxygenase and reductase domains of iNOS to understand how they form and stabilize the active dimeric enzyme. The dimeric oxygenase domain dissociated into folded, heme-containing monomers when incubated with 2-5 M urea, whereas the reductase domain unfolded under these conditions and lost its ability to catalyze NADPH-dependent cytochrome c reduction. Spectral analysis of the dissociation reaction showed that it caused structural changes within the oxygenase domain and exposed the distal side of the heme to solvent, enabling it to bind dithiothreitol as a sixth ligand. Importantly, the oxygenase domain monomers could reassociate into a dimeric form even in the absence of the reductase domain. The reaction required L-arginine and H4biopterin and completely reversed the structural changes in heme pocket and protein structure that occurred upon dissociating the original dimer. Together, this confirms that the oxygenase domain contains all of the determinants needed for subunit dimerization and indicates that the dimeric structure greatly affects the heme and protein environment in the oxygenase domain.
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PMID:Domains of macrophage N(O) synthase have divergent roles in forming and stabilizing the active dimeric enzyme. 863 74

The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.
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PMID:Conformational requirements of a recombinant ferredoxin-NADP+ reductase precursor for efficient binding to and import into isolated chloroplasts. 866 37

The enzyme NADH oxidase (EC 1.6.99.3) has been isolated from the two thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized. In both organisms the enzyme oxidizes specifically beta-NADH in the presence of molecular oxygen and requires the presence of a flavin cofactor, showing a high specificity for FAD. A stoicheiometric amount of hydrogen peroxide to NADH is formed as the end product of the reaction, indicating that both enzymes are two-electron donors. The purified enzymes exhibit quite different molecular properties. S. acidocaldarius NADH oxidase is a monomeric protein with an estimated molecular mass of about 27 kDa, whereas S. solfataricus NADH oxidase is a dimeric protein with a molecular mass of 35 kDa per subunit; S. solfataricus NADH oxidase is purified as an FAD-containing protein, whereas S. acidocaldarius NADH oxidase does not contain a flavin molecule. Furthermore, a comparison of the N-terminal amino acid sequence shows no similarities either between the two proteins or to any other NADH oxidases. Both enzymes are essentially thermophilic. In the temperature range 20-80 degrees C, the energy of activation is almost the same for both activities, suggesting that similar energetic parameters are required. Also both oxidases display a great stability to heat. The half-life of heat inactivation is about 180 min at 90 degrees C for S. acidocaldarius NADH oxidase and 77 min at 98 degrees C for the S. solfataricus enzyme. The activity of the two enzymes is inhibited by urea and guanidine and are regulated very differently by several organic solvents.
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PMID:Purification and characterization of NADH oxidase from the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus. 886 96

Electron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (alpha and beta, molecular weights of 33,000 and 29,000) and two small molecules, FAD and AMP. In this study, in vitro refolding and unfolding of the subunits of ETF were carried out with urea as the denaturing reagent. The refolding reaction of alpha and beta was revealed to proceed kinetically in two steps: D in equilibrium with I-->N, where D,I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of alpha and beta, described below, are consistent with the concept of the so-called "molten globule state," which is frequently observed in protein refolding. (i) The conversion between D and I was very rapid. (ii) The I form showed as much secondary structure as the N form as judged from the far-UV circular dichroism. (iii) The solvent accessibility of the I form, estimated by the analysis of equilibrium unfolding experiments, was intermediate between those of the D and N forms. (iv) The standard free energy of the I form is almost the same as that of the D form. The refolding reaction progressed more slowly and the environment of the tryptophan chromophore was changed more drastically in beta refolding that in alpha refolding. We previously reported that the reconstitution of holoETF from denatured subunits is speeded up by increasing the AMP concentration. In this study, the effects of AMP, FAD, and the other subunit on the single subunit folding were examined, but no effect was detected. This result suggests that AMP plays a role in a later process, namely, assembly of the four components (refolded alpha and beta, FAD, and AMP).
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PMID:In vitro refolding and unfolding of subunits of electron-transferring flavoprotein: characterization of the folding intermediates and the effects of FAD and AMP on the folding reaction. 888 11

The cytosolic and two recombinant precursors, containing 10 and 30 amino acid spacers between the transit peptide and the mature region of the chloroplast flavoprotein ferredoxin-NADP+ reductase (FNR), were expressed in Escherichia coli cells. These proteins were purified rendering fully active precursors that contained bound FAD. Neither the transit peptide nor the spacers affected the formation of the tightly folded enzyme structure. Protease treatment of the folded precursors resulted in a rapid removal of the transit sequence, rendering an enzymatically active resistant core, even at high protease concentration. All three preproteins could be efficiently imported by isolated pea chloroplasts. Addition of the enzyme substrate NADP+ to the import medium slightly decreased the polypeptide translocation. The precursor bound to isolated chloroplasts in the presence or absence of leaf extracts was as resistant to proteolysis as the folded precursor in solution. In contrast, the FNR precursor unfolded by urea was rapidly digested even at the lowest protease concentration. Together, our results indicate that precursor unfolding may take place during translocation but not during binding to chloroplast envelopes or by interaction with leaf extract soluble factors, and that this process is independent of the distance between the transit peptide and the folded mature region of the protein.
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PMID:A fully active FAD-containing precursor remains folded up to its translocation across the chloroplast membranes. 957 70

Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P. M. Horowitz, and B. S. S. Masters (1995) Arch. Biochem. Biophys. 316, 267-274]. As in the case of CPR, FMN was relatively loosely bound to nNOS and the flavoprotein module, but FAD remained bound at concentrations of up to 2 M urea Protein fluorescence increased progressively with increasing urea concentration, but could not be correlated with changes in flavin binding. NADPH-cytochrome c reductase activity of both nNOS and the flavoprotein module, but not that of CPR, was stimulated at early time points by both urea and guanidine hydrochloride (GnHCl), with levels of initial activity returning to baseline values within 60 min after addition of the chaotropic agent. Thus, at 3-4 M urea, enhancements of reductase activities of 20- and 5-fold with nNOS and the flavoprotein module, respectively, were obtained. Comparable enhancements of 12- and 6- to 7-fold, respectively, were obtained with calmodulin (CaM)/ CaCl2 and 0.5 M GnHCl. Thus, the effects of urea and GnHCl mimicked the stimulating effects of CaM. Separate preincubations of nNOS and cytochrome c with urea or GnHCl prior to initiation of the reductase assay showed that sensitivity to chaotropic agent under these conditions was a property of nNOS and not of cytochrome c. Moreover, when the nonprotein electron acceptor 2,6-dichlorophenolindophenol was employed in place of cytochrome c, comparable stimulation of reductase activity was observed in the presence of either urea or GnHCl. Fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfate in the presence of either nNOS or the flavoprotein module was increased optimally between 3 and 4 M urea, consistent with simultaneous exposure of hydrophobic regions of both proteins to solvent and optimization of reductase activity. FMN release from nNOS, but not from the flavoprotein module, was enhanced by CaM. Addition of FMN or FMN + FAD to nNOS, in the presence or absence of urea, brought about a doubling of initial cytochrome c reductase activity, but did not prevent the eventual decline in activity to basal levels. These data are consistent with conformational changes which favor increased electron transfer similar to that achieved with nNOS in the presence of CaM.
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PMID:The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins. 970 Oct 43

Oxalate oxidase (OXO) was chemically modified using amino acid-specific reagents. The modification reactions were monitored spectrophotometrically, to follow the progress of labeling, and catalytically, to assess the effect of labeling on the enzyme function. The enzyme does not bear arginines essential for activity, since 2,3-butanedione and cyclohexanodione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Incubation of urea-pretreated OXO with N-acetylimidazole leads to labeling all 10 tyrosines without affecting the enzyme activity, thus suggesting that OXO does not have tyrosines essential for activity. However, OXO modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide followed by kinetic analysis, leads to the conclusion that the enzyme possesses one carboxylate essential for activity. When using the modifier 2,4, 6-trinitrobenzene sulfonic acid (TNBS), while 28 of the total 45 lysines are labeled within 3 h (the first 5 reacting lysines of the homopentametic enzyme are modified at a faster rate than the others), the enzyme rapidly loses 90% of its activity in the first 2 min, a period during which only one lysine is being labeled. Complete enzyme inactivation with TNBS is observed after approximately 8 min, when 5 lysines are being labeled. The modification of the first lysine also triggers the dissociation of native OXO to its subunits (after SDS-PAGE analysis), a phenomenon not observed with the other modifiers. These findings indicate that OXO bears a lysine per monomer, essential for enzyme activity. When using 5, 5-dithio-bis-(2-nitrobenzoic)acid to determine the number of disulfide bonds, in the presence of NaBH4, 10 sulfhydryls are determined, but in the absence of reducing agent, none are determined. Further, chloro-mercuribenzoate does not inactivate OXO but beta-mercaptoethanol does. Therefore, the sulfhydryls in OXO are not free but form disulfide bonds essential for activity. Furthermore, the metallo-chelating agents HgCl2 and 8-hydroxychinolin inactivate the enzyme, suggesting that barley root oxalate oxidase is a metalloenzyme. It is possible that the metal(s) are involved in the oxidative mechanism since the enzyme does not bear prosthetic groups such as FAD and FMN.
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PMID:Chemical modification of barley root oxalate oxidase shows the presence of a lysine, a carboxylate, and disulfides, essential for enzyme activity. 970 1

Enzymes from extreme halophiles have potential as catalysts in biotransformations. We have developed methods for the expression in Escherichia coli and purification of two enzymes from Haloferax volcanii: dihydrolipoamide dehydrogenase and citrate synthase. Both enzymes were expressed in E. coli using the cytoplasmic expression vectors, pET3a and pET3d. Citrate synthase was soluble and inactive, whereas dihydrolipoamide dehydrogenase was expressed as inclusion bodies. Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 microM FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH. Maximal activity was obtained after 3 days incubation at 4 degrees C. Purification of the two active enzymes was carried out using high-resolution methods. Dihydrolipoamide dehydrogenase was purified using copper-based metal ion affinity chromatography in the presence of 2 M KCl. Citrate synthase was recovered using dye-affinity chromatography in the presence of salt. A high yield of active enzyme was obtained in both cases. Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependence were comparable to those of the native enzymes. Expression of active protein was attempted both by growth of E. coli in the presence of salt and betaine, and also by using periplasmic expression vectors in combination with a high salt growth media. Neither strategy was successful.
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PMID:Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli. 1039 37

A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M(r) 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 degrees C in the absence or presence, respectively, of 8 M urea. The enzyme did not show any reductase activity. The SsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative betaalphabeta-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47-54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.
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PMID:A NAD(P)H oxidase isolated from the archaeon Sulfolobus solfataricus is not homologous with another NADH oxidase present in the same microorganism. Biochemical characterization of the enzyme and cloning of the encoding gene. 1062 24

We recently described that melatonin and some kynurenines modulate the N-methyl-D-aspartate-dependent excitatory response in rat striatal neurons, an effect that could be related to their inhibition of nNOS. In this report, we studied the effect of melatonin and these kynurenines on nNOS activity in both rat striatal homogenate and purified rat brain nNOS. In homogenates of rat striatum, melatonin inhibits nNOS activity, whereas synthetic kynurenines act in a structure-related manner. Kynurenines carrying an NH(2) group in their benzenic ring (NH(2)-kynurenines) inhibit nNOS activity more strongly than melatonin itself. However, kynurenines lacking the NH(2) group or with this group blocked do not affect enzyme activity. Kinetic analysis shows that melatonin and NH(2)-kynurenines behave as noncompetitive inhibitors of nNOS. Using purified rat brain nNOS, we show that the inhibitory effect of melatonin and NH(2)-kynurenines on the enzyme activity diminishes with increasing amounts of calmodulin in the incubation medium. However, changes in other nNOS cofactors such as FAD or H(4)-biopterin, do not modify the drugs' response. These data suggest that calmodulin may be involved in the nNOS inhibition by these compounds. Studies with urea-polyacrylamide gel electrophoresis further support an interaction between melatonin and NH(2)-kynurenines, but not kynurenines lacking the NH(2) group, with Ca(2+)-calmodulin yielding Ca(2+)-calmodulin-drug complexes that prevent nNOS activation. The results show that calmodulin is a target involved in the intracellular effects of melatonin and some melatonin-related kynurenines that may account, at least in part, for the neuroprotective properties of these compounds.
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PMID:Structure-related inhibition of calmodulin-dependent neuronal nitric-oxide synthase activity by melatonin and synthetic kynurenines. 1104 43


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