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)

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
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PMID:Thiol modification and site directed mutagenesis of the flavin domain of spinach NADH:nitrate reductase. 866 Jun 90

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH. The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group. It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies. A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring. Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine. kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme. Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified. This suggests that serine 49 is involved in the binding of the isoalloxazine ring. Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation. This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction. In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor. Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.
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PMID:Is the NAD(P)H:flavin oxidoreductase from Escherichia coli a member of the ferredoxin-NADP+ reductase family?. Evidence for the catalytic role of serine 49 residue. 866 85

Inactivation of D-amino acid oxidase occurred by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu2+ and Hg2+. It was also sensitive to oxidative inactivation by Fe2+ and H2O2 when both reagents were added in millimolar amounts. When oxidatively inactivated D-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion of D-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of the D-amino acid oxidase activity. Treatment of apo-D-amino acid oxidase from T. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.
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PMID:Studies on the inactivation of the flavoprotein D-amino acid oxidase from Trigonopsis variabilis. 873 70

As part of an ongoing search for susceptibility loci for NIDDM, we tested 19 genes whose products are implicated in insulin secretion or action for linkage with NIDDM. Loci included the G-protein-coupled inwardly rectifying potassium channels expressed in beta-cells (KCNJ3 and KCNJ7), glucagon (GCG), glucokinase regulatory protein (GCKR), glucagon-like peptide I receptor (GLP1R), LIM/homeodomain islet-1 (ISL1), caudal-type homeodomain 3 (CDX3), proprotein convertase 2 (PCSK2), cholecystokinin B receptor (CCKBR), hexokinase 1 (HK1), hexokinase 2 (HK2), mitochondrial FAD-glycerophosphate dehydrogenase (GPD2), liver and muscle forms of pyruvate kinase (PKL, PKM), fatty acid-binding protein 2 (FABP2), hepatic phosphofructokinase (PFKL), protein serine/threonine phosphatase 1 beta (PPP1CB), and low-density lipoprotein receptor (LDLR). Additionally, we tested the histidine-rich calcium locus (HRC) on chromosome 19q. All regions were tested for linkage with microsatellite markers in 751 individuals from 172 families with at least two patients with overt NIDDM (according to World Health Organization criteria) in the sibship, using nonparametric methods. These 172 families comprise 352 possible affected sib pairs with overt NIDDM or 621 possible affected sib pairs defined as having a fasting plasma glucose value of >6.1 mmol/l or a glucose value of >7.8 mmol/l 2 h after oral glucose load. No evidence for linkage was found with any of the 19 candidate genes and NIDDM in our population by nonparametric methods, suggesting that those genes are not major contributors to the pathogenesis of NIDDM. However, some evidence for suggestive linkage was found between a more severe form of NIDDM, defined as overt NIDDM diagnosed before 45 years of age, and the CCKBR locus (11p15.4; P = 0.004). Analyses of six additional markers spanning 27 cM on chromosome 11p confirmed the suggestive linkage in this region. Whether an NIDDM susceptibility gene lies on chromosome 11p in our population must be determined by further analyses.
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PMID:Genetics of NIDDM in France: studies with 19 candidate genes in affected sib pairs. 916 80

1. The construction of three-dimensional models of CYP2B isozymes from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6), based on a multiple sequence alignment with CYP102, a unique eukaryotic-like bacterial P450 (in terms of possessing an NADPH-dependent FAD- and FMN-containing oxidoreductase redox partner) of known crystal structure, is reported. 2. The enzyme models described are shown to be consistent with experimental evidence from site-directed mutagenesis studies, antibody recognition sites and amino acid residues identified as being associated with redox partner interactions, together with the location of a key serine residue (Ser-128) likely to be involved in protein kinaseA-mediated phosphorylation. 3. A substantial number of known substrates and inhibitors of CYP2B isozymes are shown to fit the putative active sites of the enzyme models in agreement with their reported position of metabolism or mode of inhibition respectively. In particular, there is complementarity between the characteristic non-planar geometries of CYP2B substrates and key groups in the enzymes' active sites. 4. Molecular modelling of CYP2B isozymes appears to rationalize a number of the reported findings from quantitative structure-activity relationship investigations on series of CYP2B substrates and inhibitors.
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PMID:Molecular modelling of mammalian CYP2B isoforms and their interaction with substrates, inhibitors and redox partners. 917 87

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.
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PMID:The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior. 938 Jun 71

The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacterium Synechocystis sp. strain PCC 6803. In the present work the gene (lpdA; database accession number Z48564) encoding the apoprotein of this LPD in Synechocystis PCC 6803 has been identified, sequenced and analysed. The lpdA gene codes for a protein starting with methionine, which is post-translationally removed. The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD). The LPD is an acidic protein with a calculated isoelectric point of 5.17. Comparison of the amino acid sequence of the Synechocystis LPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published. As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-free Synechocystis mutant by insertionally inactivating the lpdA gene with a kanamycin-resistance cassette. However, the selected transformants appeared to be heteroallelic, containing both the intact lpdA gene and the lpdA gene inactivated by the drug-resistance cassette. The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium. Growth over a prolonged time was only possible after an increased buffering of the medium. Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.
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PMID:Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp. strain PCC 6803. 938 33

Monoamine oxidase B (MAO B) is an integral protein of the outer mitochondrial membrane that is involved in the deamination of vasoactive and neuroactive amines. The oxidation of these amine substrates requires the cofactor FAD, which is covalently bound to Cys-397 of human MAO B. Previously, Glu-34 and Tyr-44 of MAO B have been identified as residues which engage in noncovalent interactions with FAD that are required for subsequent covalent FAD binding and generation of catalytic activity. In this study, we have identified two additional residues, Arg-42 and Thr-45, which form noncovalent contacts with FAD that are prerequisite steps to the covalent attachment of FAD. Arg-42 and Thr-45, along with Tyr-44, comprise part of a highly conserved flavin binding sequence, RXY(T,S), that is found in other flavoproteins, several of which have well-defined X-ray crystal structures. We tested the roles of Arg-42 and Thr-45 in MAO B by constructing mutant MAO B cDNAs which encode amino acid substitutions at these residues and expressed the variant proteins in COS-7 cells. Substitution of Arg-42 or Thr-45 with alanine resulted in complete loss of MAO B activity and FAD incorporation. However, conservative substitutions of Arg-42 with lysine or Thr-45 with serine resulted in MAO B variants that retain both partial activity and partial FAD incorporation. These results indicate that Arg-42 and Thr-45 form critical noncovalent interactions with FAD that are required for the subsequent activation of MAO B by covalent coupling of FAD.
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PMID:Arginine-42 and threonine-45 are required for FAD incorporation and catalytic activity in human monoamine oxidase B. 972 50

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.
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PMID:Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain. 1002 81

It is well established that vascular plants, diatoms, and some species of marine algal flagellates have acquired an absolute requirement for boron (B), although the primary role remains unknown. Discovery of naturally occurring organoboron compounds, all ionophoric macrodiolide antibiotics with a single B atom critical for activity, established at least one biochemical role of B. The unusual nature of B chemistry suggests the possibility of a variety of biological roles for B. At physiological concentrations and pH, B may react with one N group or one to four hydroxyl groups on specific biological ligands with suitable configuration and charge to form dissociable organoboron compounds or complexes. Suitable ligands include pyridine (e.g., NAD+ or NADP) or flavin (e.g., FAD) nucleotides and serine proteases (SP). B reacts with the cis adjacent hydroxyls on the ribosyl moiety of the nucleotides or, in the serine proteases, the N on the imidazole group of histidine or the hydroxyl group on the serine moiety. Reversible inhibition by B of activity of SP or oxidoreductases that require pyridine or flavin nucleotides is well known. Therefore, a proposed essential role for B is as a regulator of relevant pathways, including respiratory burst, that utilize these enzymes.
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PMID:Regulation of enzymatic activity: one possible role of dietary boron in higher animals and humans. 1005 Sep 21


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