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)

We studied the roles of Thr-136 (T136) and Glu-137 (E137) in the biogenesis of medium chain acyl-CoA dehydrogenase (MCAD) by altering the former to Ser (T136S), Asp (T136D), or Leu (T136L) and the latter to Asp (E137D), Gln (E137Q), or Lys (E137K). After import into mitochondria, T136S and E137D were assembled into the native tetramer as efficiently as the wild-type. The tetrameric assembly of four other variants with a nonconservative substitution was severely impaired. When expressed in Escherichia coli as the mature subunit, the amounts of the catalytically active forms of T136S and E137D were comparable to wild-type, whereas four nonconservative variants were lost as aggregates. Of these nonconservative variants, only T136D formed catalytically active tetramer when the culture broth and buffers were supplemented with riboflavin and FAD, respectively. Culturing T136L or E137K at a lower temperature (28 degreesC) did not increase the yield at all, suggesting the severity of disruption of biogenesis. These results, together with the previous crystallographic findings, indicate that the T136 hydroxyl is a major FAD-binding site, and that E137 carboxyl plays a key role in the beta-domain folding, through salt bridge formation with K164. These findings also support the notion that the isoalloxazine ring plays a critical role in the MCAD folding, presumably exerting nucleating effects.
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PMID:The roles of threonine-136 and glutamate-137 of human medium chain acyl-CoA dehydrogenase in FAD binding and peptide folding using site-directed mutagenesis: creation of an FAD-dependent mutant, T136D. 975 Jan 63

In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners.
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PMID:Complex formation between Azotobacter vinelandii ferredoxin I and its physiological electron donor NADPH-ferredoxin reductase. 991 36

Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest kcat whereas the yeast reductase gave the best kcat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activities with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.
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PMID:Differential redox and electron-transfer properties of purified yeast, plant and human NADPH-cytochrome P-450 reductases highly modulate cytochrome P-450 activities. 999 Mar 23

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

The digestive gland and other tissues of several species of terrestrial gastropod mollusc contain an aliphatic alcohol oxidase activity (EC1.1.3.13). The enzyme is FAD dependent, consumes oxygen and generates hydrogen peroxide and the corresponding aldehyde. Saturated primary alcohols are favoured as substrates with octanol preferred with an apparent Km of 3-4 microM. The activity is clearly distinguishable from previously reported molluscan aromatic alcohol oxidase (EC1.1.3.7) on the basis of FAD dependence, sensitivity to heat treatment and high salt concentration and with regard to substrate preferences. The aliphatic alcohol oxidase is membrane associated and most likely localised to the endoplasmic reticulum. Extraction of membranes with 1% Igipal solubilises the enzyme in active form. This enzyme is a further example of an oxidase apparently restricted to molluscs.
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PMID:Gastropod mollusc aliphatic alcohol oxidase: subcellular localisation and properties. 1090 67

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NO&z.rad; production in biological systems, controversy still remains concerning whether NOS directly produces NO&z.rad;. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO(-)), which is then converted to NO&z.rad; by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NO&z.rad; from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NO&z.rad; measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NO&z.rad; from NO(-) (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe(2+)-MGD, NO&z.rad; generated characteristic triplet NO&z.rad;-Fe(2+)-MGD signals (g = 2. 04, a(N) = 12.7 G), whereas NO(-) from Angeli's salt was EPR silent. Both NO&z.rad; and NO(-) gave rise to NO&z.rad;-Fe(2+)-MGD signals when Fe(3+)-MGD was used. Strong NO&z.rad; signals were measured from purified nNOS using the NO&z.rad; selective Fe(2+)-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NO&z.rad; and NO(-) and this depends on the redox status of the iron. The detection of NO&z.rad; from purified NOS by Fe(2+)-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NO&z.rad; but not NO(-).
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PMID:Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase. 1105 82

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.
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PMID:Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae. 1110 87

The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II. Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA. We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue. MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family). Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD. In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein. HFCD enzymes are characterized by a conserved His residue which is part of the active site. Exchange of this His residue for Asn led to inactivation of MrsD. The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities.
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PMID:The flavoprotein MrsD catalyzes the oxidative decarboxylation reaction involved in formation of the peptidoglycan biosynthesis inhibitor mersacidin. 1184 51

Adrenodoxin reductase (AR) and adrenodoxin (Adx) are components of the mammalian mitochondrial steroid-hydroxylating system. Crystal structures of Adx, AR and a cross-linked Adx-AR complex have recently been determined. Based on these, we have carried out a modeling and docking study to characterize the recognition between AR, Adx and cytochrome c (Cytc). To rationalize the recognition process, electrostatic potentials were calculated by solving the Poisson-Boltzmann equations. In the Adx-AR complex modeled, a negatively charged surface of Adx recognizes a positive surface of AR, as in the crystal structure of the Adx-AR complex, proving the correct parameterization for the energy calculations. After forming salt bridges between the polar primary binding sites of Adx and AR, charge compensation causes a domain movement in AR, which closes the binding cleft by 2-4 A. Thereby, a secondary polar binding site is closed and the electron transfer pathways between the FAD of AR and the [2Fe-2S] cluster of Adx are adjusted. Next, the model structure of a complex between Adx and Cytc was derived. The lowest-energy complex between Adx and Cytc matches earlier chemical modification and cross-linking experiments, which proposed polar interactions of Lys13, Lys27, Lys72 and Lys79 of Cytc with acidic residues in Adx. Because of the short distance of 9.4 A between the redox centers, a complex, productive in electron transfer via a different outlet pathway from the inlet route in Adx, is expected. However, a ternary complex cannot be formed between the Adx-AR complex and Cytc because of steric hindrance. Therefore, a shuttle model for the role of Adx in the electron transfer process to Cytc is preferable to a relay model. In addition, no preferable docking site could be detected for a second Adx when probing the Adx-AR complex, which is required for a quaternary organized-cluster model of all redox partners of the hydroxylase system.
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PMID:Modeling of electrostatic recognition processes in the mammalian mitochondrial steroid hydroxylase system. 1264 71

The three-dimensional structures of K72E, K75R, K75S, K75Q, and K75E Anabaena Ferredoxin-NADP+ reductase (FNR) mutants have been solved, and particular structural details of these mutants have been used to assess the role played by residues 72 and 75 in optimal complex formation and electron transfer (ET) between FNR and its protein redox partners Ferredoxin (Fd) and Flavodoxin (Fld). Additionally, because there is no structural information available on the interaction between FNR and Fld, a model for the FNR:Fld complex has also been produced based on the previously reported crystal structures and on that of the rat Cytochrome P450 reductase (CPR), onto which FNR and Fld have been structurally aligned, and those reported for the Anabaena and maize FNR:Fd complexes. The model suggests putative electrostatic and hydrophobic interactions between residues on the FNR and Fld surfaces at the complex interface and provides an adequate orientation and distance between the FAD and FMN redox centers for efficient ET without the presence of any other molecule as electron carrier. Thus, the models now available for the FNR:Fd and FNR:Fld interactions and the structures presented here for the mutants at K72 and K75 in Anabaena FNR have been evaluated in light of previous biochemical data. These structures confirm the key participation of residue K75 and K72 in complex formation with both Fd and Fld. The drastic effect in FNR activity produced by replacement of K75 by Glu in the K75E FNR variant is explained not only by the observed changes in the charge distribution on the surface of the K75E FNR mutant, but also by the formation of a salt bridge interaction between E75 and K72 that simultaneously "neutralizes" two essential positive charged side chains for Fld/Fd recognition.
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PMID:Structural analysis of interactions for complex formation between Ferredoxin-NADP+ reductase and its protein partners. 1578 5


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