Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. It has previously been established that monoamine oxidase (MAO)-B participates in the metabolism of milacemide [2-(pentylamino)acetamide]. Furthermore, in rats, inhibition of FAD-dependent polyamine oxidase (PAO) was found to decrease the urinary excretion of two milacemide metabolites, termed UK1 and UK2. 2. Using gas chromatography-mass spectrometry, UK1 was identified as oxamic acid and UK2 as 2-hydroxyacetamide, confirming that PAO is involved in the metabolism of milacemide. 3. Thus, two FAD-dependent amine-oxidizing enzymes, MAO and PAO, contribute to the metabolism of milacemide. Milacemide appears to be the first non-polyamine xenobiotic in the metabolism of which PAO participates.
 ...
PMID:Involvement of FAD-dependent polyamine oxidase in the metabolism of milacemide in the rat. 163 8

Flavin-containing monooxygenase (FMO; EC 1.14.13.8) was purified from mouse kidney microsomes and compared to that isolated from mouse liver microsomes. The purified enzymes from kidney and liver appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 58,000 daltons. On wide range (pH 3.5 to 9.0) isoelectric focusing, FMOs from kidney and liver resolved as a single band with an isoelectric point of 8.2. The enzymes from both kidney and liver have a pH optimum of 9.2. Thiobenzamide-S-oxidation catalyzed by both enzymes was sensitive to inhibition by the competitive inhibitors thiourea and methimazole. At an n-octylamine concentration of 3 mM, thiobenzamide-S-oxidation by the kidney FMO was increased by 122% and that by the liver FMO by 148%. Km and Vmax values were determined and compared between the two tissue enzymes for xenobiotic substrates containing nucleophilic nitrogen, sulfur or phosphorus atoms. In general, for most FMO substrates, Km and Vmax values were similar between kidney and liver FMO with only a few exceptions. The Km and Vmax values for fenthion for kidney were only half of those observed for liver FMO. Fonofos was unusual in having a low Km as well as a low Vmax for both tissue enzymes. Anti-sera developed to the FMO purified from kidney and liver showed cross-reactivity with each purified enzyme as well as with a protein with the same molecular weight as the purified FMO present in both kidney and liver microsomes. These bands showed equal intensity based on an equivalent amount of protein. Analysis of kidney and liver FMO by proteolytic digestion followed by visualization of peptides by silver staining or immunoblotting showed only minor differences between the enzymes of the two tissues. The amino acid composition of both mouse kidney and liver FMO was low in methionine and histidine and rich in aspartate/asparagine, glutamate/glutamine, leucine, valine and glycine. Edman degradation of the purified mouse kidney and liver FMO provided a single amino acid sequence of the NH2-terminus. This sequence matched exactly with the cDNA-deduced sequence reported for the pig and rabbit liver beginning with the fifth amino acid and contained the highly conserved FAD-binding domain Gly-X-Gly-X-X-Gly, commonly found in a number of other FAD-binding proteins. These studies indicate that the renal and hepatic forms of FMO from mouse are similar enzymes that are immunologically related and show only a few minor differences.
 ...
PMID:The flavin-containing monooxygenase of mouse kidney. A comparison with the liver enzyme. 193 Feb 64

Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing mono-oxygenase and the stoichiometry between substrate, O2 and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.
 ...
PMID:The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450. 650 63

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397.
 ...
PMID:Flavinylation of monoamine oxidase B. 755 33

Monoamine oxidase (MAO)-A and MAO-B are FAD-containing mitochondrial enzymes which catabolize biogenic and xenobiotic amines. The N-terminal regions of both forms of MAO contain an ADP-binding consensus sequence found in several dinucleotide-dependent enzymes, but otherwise show remarkable sequence differences. In order to investigate whether the N-terminal region of MAOs participates in the different catalytic properties and inhibitor specificities exhibited by MAO-A and MAO-B, we constructed chimeric A/B forms and expressed them in a human embryonic kidney cell line (293 cells). The MAO-A chimeric form containing the N-terminus (36 amino acids) of MAO-B and the B chimera having the first 45 amino acid sequence of MAO-A were both catalytically active. Compared to the respective wild-type form, they did not show any significant difference in their catalytic properties (Km, kcat) towards the substrates tested or in their sensitivity towards inhibitors. This indicates that the N-terminal region of the two isoenzymes is not involved in the different specificities of MAO-A and MAO-B. Substitution of Cys-397 of MAO-B, i.e. the residue covalently anchoring FAD, with an Ala or a His residue resulted in the total loss of enzymatic activity, suggesting that the covalent coupling of FAD to MAO occurs specifically at the-SH group of cysteine.
 ...
PMID:Characterisation of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line. 842 24

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.
 ...
PMID:Molecular cloning and kinetic characterization of a flavin-containing monooxygenase from Saccharomyces cerevisiae. 895 74

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.
 ...
PMID:Characterization of a highly conserved FAD-binding site in human monoamine oxidase B. 961 88

The flavin-containing monooxygenase (FMO) gene family is conserved and ancient with representatives present in almost all phyla so far examined. The genes encode FAD-, NADP- and O(2)-dependent enzymes that catalyse oxygenation of soft-nucleophilic heteroatom centres in a range of substrates. Although usually classified as xenobiotic-metabolising enzymes, examples of FMOs exist that have evolved to metabolise specific endogenous substrates as part of a discrete physiological process. The genome of Caenorhabditis elegans contains five predicted genes encoding putative homologs of mammalian FMOs, K08C7.2, K08C7.5, Y39A1A.19, F53F4.5 and H24K24.5, which we have named fmo and numbered fmo-1 to fmo-5, respectively. As a first step towards determining their functional role(s), we have experimentally characterised these C. elegans fmo genes including analysing reporter gene expression patterns and RNAi phenotypes. Two major gene expression patterns were observed, either intestinal or hypodermal, but no gross RNAi phenotypes were found possibly due to functional redundancy. The internal structures of fmo-2, fmo-3 and fmo-4 have been compared with orthologs identified in the related nematode C. briggsae. For each orthologous pair, a global comparison of the paired upstream intergenic regions was performed and a number of conserved noncoding sequences, which may represent potential cis-regulatory elements, identified. Phylogenetic analysis reveals that several of the fmo homologs are the result of gene duplication along the lineage leading to the nematodes.
 ...
PMID:The fmo genes of Caenorhabditis elegans and C. briggsae: characterisation, gene expression and comparative genomic analysis. 1571 98

The flavin-containing monooxygenases (FMOs) are important for xenobiotic metabolism. FMO3, the predominant FMO enzyme in human adult liver, exhibits significant interindividual variation that is poorly understood. This study was designed to identify common FMO3 genetic variants and determine their potential for contributing to interindividual differences in FMO3 expression. FMO3 single nucleotide polymorphism (SNP) discovery was accomplished by resequencing DNA samples from the Coriell Polymorphism Discovery Resource. Population-specific SNP frequencies were determined by multiplexed, single-base extension using DNA from 201 Hispanic American (Mexican descent), 201 African American, and 200 White (northern European descent) subjects. Haplotypes were inferred and population frequencies estimated using PHASE version 2.1. Multiple site-directed mutagenesis was used to introduce inferred upstream haplotypes into an FMO3/luciferase construct for functional analysis in HepG2 cells. Sequence analysis revealed seven FMO3 upstream SNPs, 11 exon SNPs, and 22 intron SNPs. Five of the latter fell within consensus splice sites. A g.72G>T variant (E24D) is predicted to impact the structure of the Rossmann fold involved in FAD binding, whereas a g.11177C>A variant (N61K) is predicted to disrupt the secondary structure of a conserved membrane interaction domain. Seven common (>1%) promoter region haplotypes were inferred in one or more of the study populations that differed in estimated frequency among the groups. Haplotype 2 resulted in an 8-fold increase in promoter activity, whereas haplotypes 8 and 15 exhibited a near complete loss of activity. In conclusion, FMO3 promoter haplotype variants modulate gene function and probably contribute to interindividual differences in FMO3 expression.
 ...
PMID:Discovery of novel flavin-containing monooxygenase 3 (FMO3) single nucleotide polymorphisms and functional analysis of upstream haplotype variants. 1585 76

Numerous mutations/polymorphisms of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), have been described in patients with Antley-Bixler syndrome (ABS), presenting with craniofacial dysmorphogenesis, and/or disordered steroidogenesis, exhibiting ambiguous genitalia. CYPOR is the obligate electron donor to 51 microsomal cytochromes P450 that catalyze critical steroidogenic and xenobiotic reactions, and to two heme oxygenase isoforms, among other redox partners. To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed. WT enzyme was green, due to air-stable FMN semiquinone (blue) and oxidized FAD (yellow). The ABS mutant V492E was blue-gray. Flavin analysis indicated that WT had a protein:FAD:FMN ratio of approximately 1:1:1, whereas approximately 1:0.1:0.9 was observed for V492E, which retained 9% of the WT k(cat)/K(m) in NADPH:cytochrome c reductase assays. V492E was reconstituted upon addition of FAD, post-purification, as shown by flavin analysis, activity assay, and near UV-visible CD. Both Y459H and V492E were expressed as membrane anchor-containing proteins, which also exhibited FAD deficiency. CYP4A4-catalyzed omega-hydroxylation of prostaglandin E1 was supported by WT CYPOR but not by either of the ABS mutants. Hydroxylation activity was rescued for both Y459H and V492E upon addition of FAD to the reaction. Based on these findings, decreased FAD-binding affinity is proposed as the basis of the observed loss of CYPOR function in the Y459H and V492E POR mutations in ABS.
 ...
PMID:Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase. 1699 38


1 2 Next >>