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)

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.
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PMID:Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger. 777 50

The activity of biotin synthesis from dethiobiotin was found in cell-free extracts of an Escherichia coli bioB transformant. Among the sulfur compounds tested, only S-adenosyl-L-methionine (AdoMet) had a significant effect, while methionine and cysteine were inert. The activity was linearly stimulated by increasing protein concentration. When the dialyzed cell-free extracts were used for the reaction, NADP+, NADPH, and FAD among the well-known cofactors tested promoted the activity. Furthermore, in the presence of AdoMet, cysteine was apparently effective for biotin synthetic activity.
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PMID:Stimulatory factors for enzymatic biotin synthesis from dethiobiotin in cell-free extracts of Escherichia coli. 778 12

The flavoprotein NADH peroxidase from Enterococcus faecalis 10C1 has been shown to contain, in addition to FAD, an unusual cysteine-sulfenic acid (Cys-SOH) redox center. The non-flavin center cycles between reduced (Cys-SH) and oxidized (Cys-SOH) states, and the 2.16 A crystal structure of the non-native cysteine-sulfonic acid (Cys-SO3H) form of the wild-type peroxidase supports the proposed catalytic role of Cys42. In this study, we have employed a site-directed mutagenesis approach in which Cys42 is replaced with Ser and Ala, neither side chain of which is capable of redox activity. Reductive titrations of both C42S and C42A mutants lead directly to full FAD reduction with 1 equiv of either dithionite or NADH, consistent with elimination of the Cys-SOH center. Direct determinations of the redox potentials for the FAD/FADH2 couples yield values of -219 and -197 mV, respectively, for C42S and C42A peroxidases, indicating that the presence of Cys42-SH in the two-electron-reduced wild-type enzyme lowers the flavin potential by approximately 100 mV. Anaerobic stopped-flow analyses of the reduction of C42S and C42A peroxidases by NADH demonstrate that in both cases flavin reduction is rapid; these results are confirmed by enzyme-monitored, steady-state kinetic analyses which, in addition, give turnover numbers approximately 0.04% that of wild-type enzyme. These results are entirely consistent with the role proposed for Cys42 in the catalytic redox cycle of wild-type NADH peroxidase and indirectly support its function as a peroxidatic center in the homologous NADH oxidase.
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PMID:Analysis of the kinetic and redox properties of NADH peroxidase C42S and C42A mutants lacking the cysteine-sulfenic acid redox center. 781 35

Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. In Aspergillus nidulans, it is encoded by the niaD gene. A homologous transformation system has been used whereby a major deletion at the niiAniaD locus of the host was repaired by gene replacement. Employing site-directed mutagenesis and this transformation system, nine niaD mutants were generated carrying specific amino acid substitutions. Mutants in which alanine replaced cysteine 150, which is thought to bind the molybdenum atom of the molybdenum-pterin, and in which alanine replaced histidine 547, which putatively binds heme iron, had no detectable nitrate reductase (NAR) activity. This clearly establishes an essential catalytic role for these residues. Of the remaining mutants, all altered in the NADPH/FAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels. Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. These findings are considered in the light of predicted structural/functional roles for the altered amino acids.
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PMID:Site-directed mutagenesis of nitrate reductase from Aspergillus nidulans. Identification of some essential and some nonessential amino acids among conserved residues. 789 4

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys449) in pig apolipoamide dehydrogenase to generate a flavinylated enzyme containing covalently bound 8-(cysteinyl)FAD. Competitive behavior is observed in reconstitution reactions containing both FAD and 8-(methylsulfonyl)FAD. Covalently bound 8-(cysteinyl)FAD is shielded from solvent, as judged by spectral comparison with model 8-(alkylthio)-flavins in various solvents. Flavinylated lipoamide dehydrogenase is monomeric and catalytically inactive. Cys449 is located in the interface domain, near the active site histidine (His452). As shown previously, Cys449 is oxidized when native enzyme is treated with cupric ions. Cys449 is close to the isoalloxazine ring of FAD in native enzyme, as judged by alignment of the pig sequence with the structure of the homologous enzyme from Azotobacter vinelandii. The residue corresponding to Cys449 in A. vinlandii lipoamide dehydrogenase (Val447) is about 9 A from the carbonyl oxygen at C(2) in the pyrimidine ring of FAD. Approximation of a substituent at position 8 in FAD with Cys449 requires a 180 degrees flip of the isoalloxazine ring as compared with its orientation in the native structure. The different flavin orientation can explain the absence of dimerization and catalytic activity. Using the same method of apoenzyme preparation, noncovalent binding was observed with 8-chloroFAD, a less reactive flavin analogue. Relatively nonspecific covalent incorporation was observed with 8-chloroFAD when apoenzyme was prepared by an older method used in previous studies with this derivative [Moore, E.G., Cardemil, E., & Massey, V. (1978) J. Biol. Chem. 253, 6413-6422].
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PMID:Affinity probing of flavin binding sites. 1. Covalent attachment of 8-(methylsulfonyl)FAD to pig heart lipoamide dehydrogenase. 791 91

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate. Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase. 791 92

The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron-sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced. HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD-binding domain of pyridine-nucleotide-dependent disulfide oxidoreductases. Additionally, 110 amino acids C-terminal to the FAD-binding consensus, a short polypeptide stretch (VX2CATID) was detected which shows similarity to the region of thioredoxine reductase that contains the active-site cysteine residues (VX2CATCD). These findings suggest that HdrA harbors the site of heterodisulfide reduction and that the catalytic mechanism of the enzyme is similar to that of pyridine-nucleotide-dependent thioredoxin reductase. HdrA was additionally found to contain four copies of the sequence motif CX2CX2CX3C(P), indicating the presence of four [4Fe-4S] clusters. Two such sequence motifs were also present in HdrC (21.76 kDa), the N-terminal amino acid sequence of which showed sequence similarity to the gamma-subunit of the anaerobic glycerol-3-phosphate dehydrogenase of Escherichia coli. HdrC is therefore considered to be an electron carrier protein that contains two [4Fe-4S] clusters. HdrB (33.46 kDa) did not show sequence similarity to other known proteins, but appears to possess a C-terminal hydrophobic alpha-helix that might function as a membrane anchor. Although hdrB and hdrC are juxtaposed, these genes are not near hdrA.
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PMID:The heterodisulfide reductase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic of pyridine-nucleotide-dependent thioredoxin reductases. 792 45

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains five sulfhydryl groups per subunit. Cysteine-->serine replacements show that the thiols are not essential for catalysis. The increased dissociation constant for FAD in mutant Cys158Ser suggests that Cys158 is important for the solvation of the pyrophosphate moiety of the prosthetic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivated by mercurial compounds. Inactivation by a spin-labeled derivative of p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Incorporation of the spin label in the other Cys-->Ser mutants strongly impairs substrate binding without affecting the catalytic properties of the FAD. The results are discussed with respect to previous tentative assignments from chemical modification studies and in light of the 3-D structure of the enzyme-substrate complex.
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PMID:Selective cysteine-->serine replacements in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens allow the unambiguous assignment of Cys211 as the site of modification by spin-labeled p-chloromercuribenzoate. 793 11

The PutA protein of Escherichia coli has two enzymatic activities: proline dehydrogenase (PDH) and delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDH). It associates with the cytoplasmic membrane as PDH and P5CDH and with put control region DNA as put repressor. Reduction of the PutA flavin by proline, a PutA conformational change and association of PutA with membranes are coincident. The nucleotide base sequence of E. coli putA was determined, that of S. typhimurium putA was updated and the deduced PutA protein sequences were surveyed for catalytic domains and ligand binding sites. The two sequences were very similar (80.5% and 95% on the nucleic acid and protein levels, respectively). Residues 650 through 1130 of PutA were very similar to the sequences of P5C dehydrogenases and aldehyde dehydrogenases from both prokaryotes and eukaryotes. Glutamate 883 and cysteine 917 of PutA were conserved with the corresponding residues in P5C dehydrogenases and with those proposed to be active site residues in the aldehyde dehydrogenases. Those relationships suggest that gamma-glutamic semialdehyde, believed to equilibrate spontaneously with P5C, is the substrate for P5C dehydrogenases. Residues 340 through 590 of PutA were similar in sequence to proline dehydrogenases from Saccharomyces cerevisiae and Drosophila melanogaster. Limited similarities were also found between residues 315 through 357 of PutA and a consensus sequence near a putative active site and FAD-binding region shared by succinate dehydrogenase sequences from several organisms. Since residues 228 through 358 of PutA were similar in sequence to several serine-pyruvate aminotransferases, PutA is proposed to catalyze the hydrolysis of P5C (a Schiff's base intermediate) to gamma-glutamic semialdehyde. A carboxyl-terminal sequence that resembles a leucine zipper motif may be involved in association of PutA with put control region DNA.
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PMID:Sequence analysis identifies the proline dehydrogenase and delta 1-pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein. 796 12

A cell-free system of a bioB transformant of Bacillus sphaericus, effecting the last step of biotin biosynthesis, namely the introduction of sulfur into dethiobiotin has been recently described. S-adenosyl methionine (SAM) is absolutely necessary for activity. We show here, through experiments with [35S]SAM and [35S]Cys, that the sulfur donor is not SAM but probably cysteine (Cys) or a derivative. This finding together with the fact that NADPH and FAD are required for activity leads us to postulate some analogy between the biotin synthase system and other systems which use SAM as a source of desoxyadenosyl radical.
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PMID:On the mechanism of biotin synthase of Bacillus sphaericus. 798 99


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