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 DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG. Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins. A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either one [4Fe-4S] or two [2Fe-2S] clusters. The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues. The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1.
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PMID:Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome. 220 Jun 72

Carotenoids comprise one of the most widespread classes of pigments found in nature. The first reactions of C40 carotenoid biosynthesis proceed through common intermediates in all organisms, suggesting the evolutionary conservation of early enzymes from this pathway. We report here the nucleotide sequence of three genes from the carotenoid biosynthesis gene cluster of Erwinia herbicola, a nonphotosynthetic epiphytic bacterium, which encode homologs of the CrtB, CrtE, and CrtI proteins of Rhodobacter capsulatus, a purple nonsulfur photosynthetic bacterium. CrtB (prephytoene pyrophosphate synthase), CrtE (phytoene synthase), and CrtI (phytoene dehydrogenase) are required for the first three reactions specific to the carotenoid branch of general isoprenoid metabolism. The homologous proteins from E. herbicola and R. capsulatus show sequence identities of 41.7% for CrtI, 33.7% for CrtB, and 30.8% for CrtE. E. herbicola and R. capsulatus CrtI also display 27.2% and 27.9% sequence identity, respectively, with R. capsulatus CrtD (methoxyneurosporene dehydrogenase). All three dehydrogenases possess a hydrophobic N-terminal domain containing a putative ADP-binding beta alpha beta fold characteristic of enzymes known to bind FAD or NAD(P) cofactors. In addition, E. herbicola and R. capsulatus CrtB show 25.2% and 23.3% respective sequence identities with the protein product of pTOM5, a tomato cDNA of unknown function that is differentially expressed during fruit ripening. These data indicate the structural conservation of early carotenoid biosynthesis enzymes in evolutionarily diverse organisms.
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PMID:Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes. 226 48

An arginine biosynthetic gene cluster, argC-argJ, of the extreme thermophilic bacterium Thermus thermophilus HB27 was isolated by heterologous complementation of an Escherichia coli acetylornithinase mutant. The recombinant plasmid (pTHM1) conferred ornithine acetyltransferase activity to the E. coli host, implying that T. thermophilus uses the energetically more economic pathway for the deacetylation of acetylornithine. pTHM1 was, however, unable to complement an E. coli argA mutant and no acetylglutamate synthase activity could be detected in E. coli argA cells containing pTHM1. The T. thermophilus argJ-encoded enzyme is thus monofunctional and is unable to use acetyl-CoA to acetylate glutamate (contrary to the Bacillus stearothermophilus homologue). Alignment of several ornithine acetyltransferase amino acid sequences showed no obvious pattern that could account for this difference; however, the monofunctional enzymes proved to have shorter N-termini. Sequence analysis of the pTHM1 3.2 kb insert revealed the presence of the argC gene (encoding N-acetylglutamate-5-semialdehyde dehydrogenase) upstream of the argJ gene. Alignment of several N-acetylglutamate-5-semialdehyde dehydrogenase amino acid sequences allowed identification of two strongly conserved putative motifs for cofactor binding: a putative FAD-binding site and a motif reminiscent of the NADPH-binding fingerprint. The relationship between the amino acid content of both enzymes and thermostability is discussed and an effect of the GC content bias is indicated. Transcription of both the argC and argJ genes appeared to be vector-dependent. The argJ-encoded enzyme activity was twofold repressed by arginine in the native host and was inhibited by ornithine. Both upstream of the argC gene and downstream of the argJ gene an ORF with unknown function was found, indicating that the organization of the arginine biosynthetic genes in T. thermophilus is new.
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PMID:Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27. 949 85

The AppA protein is required for increased photosystem gene expression upon transition of the facultatively photoheterotrophic bacterium Rhodobacter sphaeroides 2.4.1 from aerobic to anaerobic photosynthetic conditions. AppA shows no obvious similarity to proteins with established function. Genetic evidence suggests that its effect is exerted through modulation of the activity of the repressor PpsR, which controls expression of multiple photosystem genes. To gain insight into the nature of AppA involvement in redox-dependent photosystem gene expression, the appA gene was overexpressed in Escherichia coli. AppA was produced as insoluble inclusion bodies. The purified inclusion bodies were found to contain FAD. By overexpressing various deletion derivatives, we were able to localize the region of AppA sufficient for FAD binding to approximately 120 amino-terminal residues. To assess the role of FAD binding in AppA function, we constructed an AppA derivative lacking the entire FAD binding domain. Surprisingly, this derivative complemented the AppA null mutant undergoing transition from aerobic to anaerobic photosynthetic growth conditions almost to the same extent as the full-length AppA protein. When the sequence of the amino-terminal portion of AppA was examined, it was shown not to contain any known flavin binding motifs. However, two open reading frames of unknown function, showing significant similarity to the amino terminus of AppA, were identified, i.e. Synechocystis sp. Srl1694 and E. coli F403. The latter gene was amplified and overexpressed in E. coli, and the partially purified F403 protein was found to contain FAD as a cofactor. We have therefore concluded that the amino terminus of AppA represents a novel FAD binding domain present in a small group of bacterial proteins. The binding of FAD by AppA may be the first clue as to how this regulatory protein is involved in redox-regulated reactions.
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PMID:AppA, a redox regulator of photosystem formation in Rhodobacter sphaeroides 2.4.1, is a flavoprotein. Identification of a novel fad binding domain. 985 73

A cadmium-hypersensitive mutant of the fission yeast Schizosaccharomyces pombe was found to accumulate abnormally high levels of sulfide. The gene required for normal regulation of sulfide levels, hmt2(+), was cloned by complementation of the cadmium-hypersensitive phenotype of the mutant. Cell fractionation and immunocytochemistry indicated that HMT2 protein is localized to mitochondria. Sequence analysis revealed homology between HMT2 and sulfide dehydrogenases from photosynthetic bacteria. HMT2 protein, produced in and purified from Escherichia coli, was soluble, bound FAD, and catalyzed the reduction of quinone (coenzyme Q2) by sulfide. HMT2 activity was also detected in isolated fission yeast mitochondria. We propose that HMT2 functions as a sulfide:quinone oxidoreductase. Homologous enzymes may be widespread in higher organisms, as sulfide-oxidizing activities have been described previously in animal mitochondria, and genes of unknown function, but with similarity to hmt2(+), are present in the genomes of flies, worms, rats, mice, and humans.
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PMID:A fission yeast gene for mitochondrial sulfide oxidation. 1022 84

The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C4-HSL and N-(3-oxo)-dodecanoyl-L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.
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PMID:Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. 1046 25

Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent two-protein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H. Q., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S., and Gravel, R. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3059-3064). In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in the in vitro assay that utilizes artificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 micromol min(-1) mg(-1) at 25 degrees C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is approximately 100-fold slower than that of P-450 reductase. The K(m) for NADPH is 2.6 +/- 0.5 microm, and the K(act) for methionine synthase reductase is 80.7 +/- 13.7 nm for NADPH-dependent activity of methionine synthase.
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PMID:Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. 1146 10

Here we present evidence that domains in soluble proteins containing either the GXXXG or GXXXA motif are stabilized by the interaction of a beta-strand with the following alpha-helix. As an example, we characterized a beta-strand-helix interaction from the FAD or NAD(P)-binding Rossmann fold. The Rossmann fold is one of the three most highly represented folds in the Protein Data Bank (PDB). A subset of the proteins that adopt the Rossmann fold also bind to nucleotide cofactors such as FAD and NAD(P) and function as oxidoreductases. These Rossmann folds can often be identified by the short amino acid sequence motif, GX(1-2)GXXG. Here, we present evidence that in addition to this sequence motif, Rossmann folds that bind FAD and NAD(P) also typically contain either GXXXG or GXXXA motifs, where the first glycyl residue of these motifs and the third glycyl residue of the GX(1-2)GXXG motif are the same residue. These two motifs appear to stabilize the Rossmann fold: the first glycyl residue of either the GXXXG or GXXXA motif contacts the carbonyl oxygen atom from the first glycyl residue of the GX(1-2)GXXG motif consistent with the formation of a C(alpha)-H cdots, three dots, centered O hydrogen bond. In addition, both the glycyl and alanyl residues of the GXXXG or GXXXA motifs form van der Waals interactions with either a valine or isoleucine residue located either seven or eight residues further back along the polypeptide chain from the first glycine of the GXXXG or GXXXA motifs. Therefore, we combine both the GX(1-2)GXXG and GXXXG/A motifs into an extended motif, V/IXGX(1-2)GXXGXXXG/A, that is more strongly indicative than previously described motifs of Rossmann folds that bind FAD or NAD(P). The V/IXGX(1-2)GXXGXXXG/A motif can be used to search genomic sequence data and to annotate the function of proteins containing the motif as oxidoreductases, including proteins of previously unknown function.
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PMID:GXXXG and GXXXA motifs stabilize FAD and NAD(P)-binding Rossmann folds through C(alpha)-H... O hydrogen bonds and van der waals interactions. 1236 99

The multifunctional PutA flavoprotein regulates proline utilization in Escherichia coli by switching from a cytosolic DNA-binding protein to a membrane-bound enzyme with proline dehydrogenase (PRODH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. The transformation of PutA from a transcriptional repressor of the proline utilization (put) regulon to a peripheral membrane associated enzyme is mediated by a proline-dependent conformational change. Previously, limited proteolysis of PutA indicated that the conformational change involves a flexible domain of unknown function (residues 141-262) which is nearby the FAD-binding and PRODH active sites (residues 263-610). Here, we extend our understanding of the proline-dependent conformational change in PutA by investigating the intrinsic Trp fluorescence spectroscopic properties of a truncated PutA protein which contains residues 86-601 (PutA86-601) and only four Trp residues. The addition of proline to wild-type PutA86-601 decreases Trp fluorescence by 36%, indicating a substantial conformational change. An apparent rate constant of 0.59 +/- 0.06 s(-)(1) was determined for the fluorescence change by stopped-flow fluorescence measurements. The limiting rate constant for proline reduction of the FAD cofactor in PutA is 133 +/- 6 s(-)(1), demonstrating that FAD reduction precedes the conformational transition observed by Trp fluorescence. The nonreducing ligand l-tetrahydro-2-furoic acid mimics the decrease in Trp fluorescence induced by proline, indicating that both FAD reduction and ligand binding contribute to the observed conformational change in PutA86-601. W194 and W211, which are located in the flexible domain, were replaced by Phe in the PutA86-601 mutants W194F, W211F, and W194F/W211F to determine which residue is involved in the observed fluorescence change. Analysis of the PutA86-601 mutants indicated that W211 is the primary molecular marker of the conformational change caused by proline. Altogether, this work shows that the switching of PutA from a transcriptional repressor to a membrane-bound protein involves W211 in a flexible domain near the PRODH active site and occurs on a time scale that is >10-fold slower than the turnover number of PutA.
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PMID:Exploring the proline-dependent conformational change in the multifunctional PutA flavoprotein by tryptophan fluorescence spectroscopy. 1615 43

The thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 is composed of a catalytic subunit (alpha), an electron transfer subunit (beta), and a small gamma subunit of unknown function. We cloned a 1428-nucleotide gene encoding the beta subunit located immediately downstream of the alpha subunit. This completes the isolation of the genes encoding the three components of the GDH complex, which are clustered very close together with the same transcription polarity in the order gammaalphabeta. The deduced beta subunit amino acid sequence contains three typical heme-binding motifs and was 44-49% identical to the cytochrome c subunits of other FAD-dependent dehydrogenase complexes. The GDHgammaalphabeta complex of B. cepacia was successfully expressed in a fully active form in Escherichia coli by co-expression with cytochrome c maturation genes. Recombinant expression of the GDH complex was also found to restore glucose-dependent respiration in a GDH mutant of E. coli.
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PMID:Cloning and functional expression of glucose dehydrogenase complex of Burkholderia cepacia in Escherichia coli. 1633


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