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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, we have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.
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PMID:cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties. 338 49

The flavoenzymes dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) contain covalently bound FAD linked via the 8 alpha-position of the isoalloxazine ring to the imidazole N(3) of a histidine residue (Cook, R. J., Misono, K. S., and Wagner, C. (1984) J. Biol. Chem. 259, 12475-12480). The flavin-peptides from tryptic digests of these two enzymes have been isolated and sequenced. Automated sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase contained 25 amino acid residues in the following sequence: Ser-Glu-Leu-Thr-Ala-Gly-Ser- Thr-Trp-His(flavin)-Ala-Ala-Gly-Leu-Thr-Thr-Tyr-Phe-His-Pro-Gly-Ile-A sn-Leu-Lys. The sequence determined for the flavin-peptide from sarcosine dehydrogenase contained 14 amino acid residues Leu-Thr-Ser-Gly-Thr-Thr-Trp-His(flavin)-Thr-Ala-Gly-Leu-Gly-Arg.
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PMID:The amino acid sequences of the flavin-peptides of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 405 29

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.
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PMID:Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties. 640 48

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.
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PMID:Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide. 643 80

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are the folate binding proteins of rat liver mitochondria. These two enzymes contain covalently bound flavin and catalyze similar oxidative demethylation reactions (Wittwer, A. J., and Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108). Flavin-peptides have been purified from these two enzymes after proteolytic digestion by trypsin and chymotrypsin. The spectral and chromatographic properties of these flavin peptides changed after treatment with nucleotide pyrophosphatase in a manner consistent with the conversion of an FAD-peptide to an FMN-peptide. The pKa for pH-dependent fluorescence quenching of the purified flavin-peptides was not affected by borohydride reduction which, in conjunction with the pKa values, indicated that the flavin was covalently linked via the 8 alpha position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Peptides from both enzymes showed histidylflavin at the N terminus. Amino acid composition and sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase was His(flavin)-Ala-Ala-Gly-Leu. Amino acid composition and N-terminal analysis suggested the sequence of the flavin-peptide of sarcosine dehydrogenase was His(flavin)-(Ala, Gly,Thr)-Leu.
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PMID:Identification of the covalently bound flavin of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 649 Jun 27

Glutathione reductase (Mr 2 x 52 500), a flavoenzyme of known three-dimensional structure, catalyses the reduction of glutathione disulfide by NADPH. This paper describes the primary structure of the FAD-binding domain which ranges from AcAla-1 to Gly-157. The three CNBr-produced fragments (69, 10 and 80 residues) of the domain were fractionated further by enzymatic and chemical methods; isolated peptides were sequenced mainly by automatic solid-phase Edman degradation. The tryptic peptides were overlapped by chymotryptic peptides. A fragment which results from cleavage at the acid-labile bond between Asp-135 and Pro-136 supplied peptides for overlapping the CNBr-produced fragments. In addition, many peptides were ordered and overlapped by computerized comparison with a complete sequence guessed from the electron density map. With one exception the computer method and the chemical alignment gave the same results. The sequence data are discussed in the light of the secondary and tertiary structure (Schulz et al. (1978) Nature (Lond.) 273, 120--124]. The 17 N-terminal residues are not visible in the electron density map. Consequently our numbering scheme differs from that of Schulz et al. by approximately 20 residues. Acetylation of the N terminus and an unusual composition of the following residues may serve to protect the loose N-terminal section of the protein against proteolysis in situ. The four cysteinyl residues of the FAD domain are of special interest. Cys-2 at the tip of the N-terminal extension is likely to be involved in the aggregation behaviour of glutathione reductase. Cys-58 and Cys-63 (formerly Cys-41 and Cys-46) represent the enzyme's redox-active dithiol. Cys-90 with its location at the twofold axis forms a disulfide bridge with Cys-90 of the other peptide chain of the enzyme. This might be related to the fact that both peptide chains contribute to each of the two active centers. In view of the interchain disulfide bridge glutathione reductase should be regarded as a monomeric protein. The sequence of the FAD-binding domain was compared with the sequence of the NADPH-binding domain of glutathione reductase using a computer program. As discussed, the scarcity of sequence similarities does not argue against the assumption that the two nucleotide-binding domains of glutathione reductase originated by gene duplication. The pyrophosphate moiety of FAD binds to a part of the polypeptide chain which in geometric structure, in topology and in sequence resembles the phosphate loops of other nucleotide-binding proteins and of flavodoxin. Using the phosphate loop as a reference, the N-terminal sequence of five flavoproteins can be aligned. The results of Williams et al. on the sequence of lipoamide dehydrogenase (EC 1.6.4.3) and our data on glutathione reductase (EC 1.6.4.2) show clearly that these two mechanistically similar enzymes possess homologous structures.
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PMID:Glutathione reductase from human erythrocytes: amino-acid sequence of the structurally known FAD-binding domain. 703 15

A second form of rat liver FMO, FMO-A, was separated and purified by chromatography on Blue Sepharose Fast flow 6. This FMO-A is different from FMO1 by antigenic properties (anti-FMO-A did not cross-react with FMO1, and reciprocally) and by catalytic properties (the Km for trimethylamine was 3.8 microM and 141.4 microM for FMO-A and FMO1, respectively; the Km for imipramine was 536 microM and 17.4 microM for FMO-A and FMO1, respectively). Furthermore, N-terminal amino sequencing revealed differences in the primary structure of these two FMOs although they both contained the highly conserved FAD-binding domain (Gly-X-Gly-X-X-Gly).
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PMID:The flavin-containing monooxygenases in rat liver: evidence for the expression of a second form different from FMO1. 762 16

A trout liver monoamine oxidase (MAO) cDNA was cloned by screening a cDNA library with a human MAO-A cDNA probe. The trout MAO cDNA encodes 499 amino acids, with a molecular mass of 56.6 kDa. The deduced amino acid sequence of trout MAO shows 70% and 71% identity with those of human MAO-A and MAO-B, respectively. Trout MAO contains the pentapeptide sequence Ser-Gly-Gly-Cys-Tyr, to which the cofactor FAD is covalently bound. Transient expression of the cDNA in COS-7 cells shows that trout MAO oxidizes both serotonin [5-hydroxytryptamine (5-HT)] and beta-phenylethylamine (PEA), unlike human MAO-A and MAO-B, which oxidize only 5-HT and PEA, respectively. The Km for 5-HT is similar for trout MAO (130 +/- 17 mM) and human MAO-A (68 +/- 4 mM). The Km for PEA is similar for trout MAO (12.5 +/- 2.0 mM) and human MAO-B (1.5 +/- 0.2 mM). When 5-HT is used as a substrate, trout MAO is more sensitive to clorgyline (IC50, 2.8 +/- 0.2 x 10(-8) M) than deprenyl (IC50, 1.0 +/- 0.1 x 10(-6) M), a result similar to the inhibition selectivity of human MAO-A. However, trout MAO is less sensitive to clorgyline than is human MAO-A (IC50, 5.8 +/- 0.1 x 10(-10) M). Trout MAO is less sensitive to deprenyl (IC50, 4.6 +/- 0.3 x 10(-7) M) than is human MAO-B (IC50, 1.4 +/- 0.1 x 10(-9) M) when PEA is used as the substrate. These results indicate that trout MAO displays substrate and inhibitor selectivities that are not identical to those of either MAO-A and -B, and it therefore represents a novel type of MAO. The structure of trout MAO will provide insights into the substrate and inhibitor selectivities of the MAOs.
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PMID:Cloning of a novel monoamine oxidase cDNA from trout liver. 780 46

We have purified a steroid-inducible 1:2-dehydrogenase from Nocardia opaca. The final enzyme preparation was purified 120-fold with a recovery of 38%. The N-terminal amino acid sequence was determined to be: Met-Gln-Asp-Trp-Thr-Ser-Glu-(Cys)-Asp-Val-Leu-Val-Val-Gly-. From the genomic library of Nocardia opaca in the plasmid pUC19, a clone designated as pSTD23 containing a 0.9 kb KpnI-PstI fragment was found to hybridize with an oligonucleotide probe corresponding to the first six amino acids from the N-terminal of the purified protein. The nucleotide sequence of the upstream region and a part of the structural domain were determined. The sequence of the first 56 amino acids of the steroid 1:2-dehydrogenase from Nocardia opaca as deduced from its gene sequence showed a 58% homology with the corresponding gene from Pseudomonas testosteroni, and the conservative sequences in the FAD-binding domain were also determined.
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PMID:Improved purification of steroid 1:2-dehydrogenase from Nocardia opaca and partial characterization of its cloned gene sequence. 791 96

A microsomal flavin-containing monooxygenase (FMO) was purified 77-fold from macacque liver microsomes on the basis of its methyl p-tolyl sulfoxidase activity. Sequential chromatography on anion- and cation-exchangers, lauryl-Sepharose and 2',5'-ADP-Sepharose provided a purified preparation which exhibited an apparent molecular mass of 59 kDa and a pI of 8.3. N-terminal amino-acid sequencing revealed the characteristic Gly-X-Gly-X-X-Gly consensus sequence for the putative FAD-binding domain of microsomal FMO. In marked contrast to the well-characterized hepatic and pulmonary forms present in experimental animals, the macacque liver enzyme displayed stereoselectivity for sulfoxidation of p-tolyl methyl sulfide on the pro-S rather than the pro-R face of the substrate. Polyclonal antibodies raised against the macacque liver form exhibited little or no cross-reactivity with major purified forms of the enzyme isolated from rabbit liver, guinea-pig liver or rabbit lung. Anti-macacque liver FMO did not cross-react with human fetal liver or adult kidney microsomes, but did recognize a 59 kDa constituent of human adult liver microsomes. The intensity of this immunoreactive 59 kDa band correlated well with human liver microsomal N,N-dimethylaniline N-oxygenase activity. We conclude that human adult liver selectively expresses a microsomal FMO which is functionally and immunochemically distinct from the FMO form(s) present in human fetal liver and adult kidney, and from the major hepatic and pulmonary forms present in common laboratory animals.
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PMID:Purification of macaque liver flavin-containing monooxygenase: a form of the enzyme related immunochemically to an isozyme expressed selectively in adult human liver. 844 76


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