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)

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.
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PMID:Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase. 1277 35

Nitrate reductase (NR; EC 1.6.6.1-3) catalyzes NAD(P)H reduction of nitrate to nitrite. NR serves plants, algae, and fungi as a central point for integration of metabolism by governing flux of reduced nitrogen by several regulatory mechanisms. The NR monomer is composed of a ~100-kD polypeptide and one each of FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT). NR has eight sequence segments: (a) N-terminal "acidic" region; (b) Mo-MPT domain with nitrate-reducing active site; (c) interface domain; (d) Hinge 1 containing serine phosphorylated in reversible activity regulation with inhibition by 14-3-3 binding protein; (e) cytochrome b domain; (f) Hinge 2; (g) FAD domain; and (h) NAD(P)H domain. The cytochrome b reductase fragment contains the active site where NAD(P)H transfers electrons to FAD. A complete three-dimensional dimeric NR structure model was built from structures of sulfite oxidase and cytochrome b reductase. Key active site residues have been investigated. NR structure, function, and regulation are now becoming understood.
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PMID:NITRATE REDUCTASE STRUCTURE, FUNCTION AND REGULATION: Bridging the Gap between Biochemistry and Physiology. 1501 11

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.
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PMID:Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production. 1510 59

Erythrocyte NADH-cytochrome b(5) reductase reduces methaemoglobin to functional haemoglobin. In order to examine the function of the enzyme, the structure of NADH-cytochrome b(5) reductase from human erythrocytes has been determined and refined by X-ray crystallography. At 1.75 A resolution, the root-mean-square deviations (r.m.s.d.) from standard bond lengths and angles are 0.006 A and 1.03 degrees , respectively. The molecular structure was compared with those of rat NADH-cytochrome b(5) reductase and corn nitrate reductase. The human reductase resembles the rat reductase in overall structure as well as in many side chains. Nevertheless, there is a large main-chain shift from the human reductase to the rat reductase or the corn reductase caused by a single-residue replacement from proline to threonine. A model of the complex between cytochrome b(5) and the human reductase has been built and compared with that of the haem-containing domain of the nitrate reductase molecule. The interaction between cytochrome b(5) and the human reductase differs from that of the nitrate reductase because of differences in the amino-acid sequences. The structures around 15 mutation sites of the human reductase have been examined for the influence of residue substitutions using the program ROTAMER. Five mutations in the FAD-binding domain seem to be related to cytochrome b(5).
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PMID:Structure of human erythrocyte NADH-cytochrome b5 reductase. 1550 98

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
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PMID:Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation. 1568 31

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.
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PMID:Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis. 1570 57

Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.
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PMID:Viscosity effects on eukaryotic nitrate reductase activity. 1589 95

Excessive nitrate accumulated in plants affects vegetable quality severely and excessive nitrate ingestion would do harm to human health. Assimilatory NADH: nitrate reductase (NR, EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. Enhancing the activity of NR is conducive to reduce the concentration of nitrate in plants. The experiments were conducted to investigate the activity of nitrate reductase in different plant tissues and the relationship between external inducing solution concentration and NR activity (NRA) in plant leaves. Six plant seedlings growing in solution culture were deprived of an external nitrogen (N) supply for 2 weeks. On selected days, three of six plant seedlings were exposed to 50mmol/L NO3- for 0, 2, 5, 8, 11h, and four of the six plant seedlings were exposed to 0, 10, 30, 50mmol/L NO3- for 2h. The NRA was determined in vivo at 538nm using spectrophotometer. The results showed that NRA increased when those plant seedlings were induced by nitrate solution. The change trends of NRA in roots and in leaves of cole, pea and tomato were different during treating time. The NRA in cole leaves was higher than that in its root and in other two plants and increased along with inducing time, but the NRA in bea and tomato was highest when the treating time was 8h and 2h, respectively. The highest NRA in leaves of three kinds of Chinese cabbages and tomato was induced by different concentrations of KNO3 solution. In tomato leaves, the highest NRA was induced by 10 - 30mmol/L KNO3 solution. In three Chinese cabbages, Brassica chinensis L. cv. AJH, XBC and KR-605, the highest NRA was induced by 10, 30, 10mmol/L KNO3 solution, respectively. The results indicated that the response manners of NRA in plants to external nitrate solutions were different. According to these results, the level of NR mRNA in plants could be enhanced by nitrate inducement. The total RNA was isolated from tomato leaves and root which induced by 30mmol/L KNO3 solution for 2h, and NR cDNA was obtained by RT-PCR using the specific primers. The fragments of PCR products were cloned and sequenced. There are 2736 base pairs in the whole cDNA fragment. The deduced protein sequence contains 911 amino acids. The NR gene can be fused to the CaMV 35S promoter, then introduced to higher plants, such as vegetables. It is hoped to decrease drastically the nitrate content of the transgenic plants.
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PMID:[Induced activity of nitrate reductase by nitrate and cloning of nitrate reductase gene]. 1596 98

NADH-cytochrome b(5) reductase deficiency results clinically in either type I or type II recessive congenital methemoglobinemia. The more severe type II form is associated with a global deficiency of cytochrome b(5) reductase and is characterized by cyanosis with neurological dysfunction. In contrast, the only symptom for type I is cyanosis. We have identified a novel G to A mutation at position 15,635 in the DIAI gene of a 4-month-old baby that results in a glycine to serine substitution at codon 75 in the cytochrome b(5) reductase protein. The G75S mutation, located in the FAD-binding lobe of cytochrome b(5) reductase, was found in association with the previously described V252M variant. The V252M mutation is present in the NADH-binding domain and associated with both types I and II recessive congenital methemoglobinemia. Since the G75S and V252M mutations represent radical changes in differing regions of cytochrome b(5) reductase, generating and characterizing these variants singly and in combination using a rat heterologous expression system would provide insight into the differences between types I and II disease at the molecular level. Although all three variants were found to retain stoichiometric levels of FAD with spectroscopic and thermodynamic properties comparable to those of native cytochrome b(5) reductase, all exhibited decreased catalytic efficiency and reduced protein stability reflecting the position of the mutations in the primary structure. The G75S variant retained only 11% of the catalytic efficiency of the wild-type enzyme. Thus, cytochrome b(5) reductase deficient patients who are heterozygous for either FAD- or NADH-binding lobe mutations can exhibit the clinically less severe type I phenotype.
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PMID:Identification and characterization of the novel FAD-binding lobe G75S mutation in cytochrome b(5) reductase: an aid to determine recessive congenital methemoglobinemia status in an infant. 1631 Mar 81

A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.
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PMID:Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum. 1734 33


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