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)

In Escherichia coli cells expressing 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoprotein with covalently bound FAD, approximately 40% of the polypeptide is in its apoform. We investigated whether in vivo holoenzyme formation was influenced by the association of the apoenzyme with cellular chaperones. Immunoprecipitation of apoenzyme-containing cell extract with protein-A-Sepharose-bound 6-HDNO- or GroEL-specific antibodies failed to reveal the formation of complexes between these proteins. The limiting factor in holoenzyme formation in vivo appeared to be the intracellular supply of phosphorylated tricarbon compounds (e.g. glycerol-3-P) acting as allosteric effectors in the flavinylation reaction. When holoenzyme formation from purified apo6-HDNO was investigated in vitro, addition of GroEL and GroES to the reaction assays increased the yield of holoenzyme formation. The observed increase in apoenzyme to holoenzyme transition was ATP independent, and the effect of GroE could be simulated by high concentrations of glycerol (40%). Apparently, a nonspecific protein-protein interaction between the GroE proteins and the apo6-HDNO favored holoenzyme formation. The refolding of guanidinium hydrochloride-unfolded holoenzyme, however, was catalyzed by GroEL and GroES in an ATP-dependent reaction. Recovery of the native, enzymatically active, conformation ranged from 30 to 40%. When apo6-HDNO was denatured and refolded, the same dependence on GroE and ATP was observed in the recovery of a conformation able to incorporate FAD and to holoenzyme. [14C] FAD in the refolding assay yielded radioactively labeled 6-HDNO demonstrating the autocatalytical covalent incorporation of FAD into the polypeptide during the folding process.
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PMID:GroE dependence of refolding and holoenzyme formation of 6-hydroxy-D-nicotine oxidase. 135 85

Neurospora crassa NAD(P)H-nitrite reductase, encoded by the nit-6 gene, is a soluble, alpha2-type homodimeric protein composed of 127-kDa polypeptide subunits. This multicenter oxidation-reduction enzyme utilizes either NADH or NADPH as electron donor and possesses as prosthetic groups two iron-sulfur (Fe4S4) clusters, two siroheme groups, and two FAD molecules. The native activity of the enzyme is the NAD(P)H-dependent reduction of nitrite to ammonia. In addition, N. crassa nitrite reductase displays several partial activities in vitro, including a siroheme-independent NAD(P)H-cytochrome c reductase activity and an FAD-independent dithionite-nitrite reductase activity. These partial activities are presumed to be manifestations of discrete functional domains within the protein. A full-length nit-6 cDNA was constructed and used in developing an expression system within E. coli capable of yielding high levels of NADPH-nitrite reductase activity. Maximal expression was obtained in nirB- E. coli cells grown anaerobically at 22 +/- 1 degrees C, in conjunction with co-expression of a plasmid-borne cysG gene (encoding the rate-limiting enzyme in siroheme synthesis) and co-transformation with plasmid pGroESL (encoding bacterial chaperonins GroES and GroEL). Dissection of gene segments encoding putative functional domains within the nit-6 gene was performed. Expression of a partial cDNA construct encoding the FAD-/NAD-binding domain yielded extracts with NADPH-cytochrome c reductase activity but no NADPH-nitrite reductase activity or dithionite-nitrite reductase activity. Expression of a cDNA construct encoding the (Fe4S4)-siroheme-binding domain resulted in extracts possessing dithionite-nitrite reductase activity but no NADPH-nitrite reductase or NADPH-cytochrome c reductase activity. Analysis of site-directed mutations altering amino acid residues Cys-331 within the FAD-/NAD-binding domain and Ser-755 within the (Fe4S4)-siroheme-binding domain of the nitrite reductase demonstrated that these residues were not essential for native or partial enzyme activity. Cys-757 within the (Fe4S4)-siroheme-binding domain was essential for native enzyme activity.
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PMID:Functional dissection and site-directed mutagenesis of the structural gene for NAD(P)H-nitrite reductase in Neurospora crassa. 879 48

An open reading frame from yeast coding for a homologue of flavin containing monooxygenase (FMO) has been cloned into several Escherichia coli expression vectors. A His10 peptide attached to the amino terminus produced a high yield of soluble protein when coexpressed with GroEL and GroES. The protein was purified on an affinity column and characterized. The protein binds one mole per mole of flavin but the binding is relatively weak and 50 microM exogenous FAD is used to maintain full occupancy. The yeast enzyme, like mammalian enzymes, exhibits NADPH oxidase activity. The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show substrate activity. The Km values for these are 7.0, 9.9, and 1.3 mM, respectively; kcat values are 94, 246, and 94 per min, respectively. The enzyme apparently does not accept xenobiotic compounds but may be involved in maintaining cellular reducing potential, probably through its action on cysteamine. This activity may represent the initial role of the FMO family of enzymes, giving rise to the multigene family of drug metabolizing enzymes seen in modern mammals.
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PMID:Molecular cloning and kinetic characterization of a flavin-containing monooxygenase from Saccharomyces cerevisiae. 895 74

Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired. Among these dehydrogenases are the four chain length-specific flavoprotein dehydrogenases of fatty acid beta-oxidation. In this investigation, two mutations in the alpha subunit that have been identified in patients were expressed in Escherichia coli. Of the two mutant alleles, alphaT266M and alphaG116R, the former is the most frequent mutation found in patients with ETF deficiency. The crystal structure of human ETF shows that alphaG116 lies in a hydrophobic pocket, under a contact residue of the alpha/beta subunit interface, and that the hydroxyl hydrogen of alphaT266 is hydrogen-bonded to N(5) of the FAD; the amide backbone hydrogen of alphaT266 is hydrogen-bonded to C(4)-O of the flavin prosthetic group (Roberts, D. L., Frerman, F. E. and Kim, J-J. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14355-14360). Stable expression of the alphaG116R ETF required coexpression of the chaperonins, GroEL and GroES. alphaG116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts. It is unstable and could not be extensively purified. The alphaT266M ETF was purified and characterized after stabilization to proteolysis in crude extracts. Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein. The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF. The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl-CoA dehydrogenases which reduce the flavin to the semiquinone. However, kcat/Km of ETF-QO in a coupled acyl-CoA:ubiquinone reductase assay with oxidized alphaT266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the alphaT266M ETF semiquinone catalyzed by ETF-QO.
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PMID:Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein. 933 18

The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.
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PMID:Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli. 1821 63