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)

Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.
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PMID:Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3. 1790 5

Transcriptional activity and chromatin structure accessibility are correlated with the methylation of specific histone residues. Lysine-specific demethylase 1 (LSD1) is the first discovered histone demethylase, which demethylates Lys4 or Lys9 of histone H3, using FAD. Among the known monoamine oxidase inhibitors, tranylcypromine (Parnate) showed the most potent inhibitory effect on LSD1. Recently, the crystal structure of LSD1 and tranylcypromine was solved at 2.75 A, revealing a five-membered ring fused to the flavin of LSD1. In this study, we refined the crystal structure of the LSD1-tranylcypromine complex to 2.25 A. The five-membered ring model did not fit completely with the electron density, giving R(work)/R(free) values of 0.226/0.254. On the other hand, the N(5) adduct gave the lowest R(work)/R(free) values of 0.218/0.248, among the tested models. These results imply that the LSD1-tranylcypromine complex is not completely composed of the five-membered adduct, but partially contains an intermediate, such as the N(5) adduct.
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PMID:Crystal structure of histone demethylase LSD1 and tranylcypromine at 2.25 A. 1803 63

The gene pgaM is involved in the biosynthesis of an angucycline-type polyketide antibiotic in Streptomyces sp. PGA64. It encodes a two-domain polypeptide consisting of an N-terminal flavoprotein oxygenase and a C-terminal short-chain alcohol dehydrogenase/reductase, which are fused together at the translational level as a result of end codon deletion. Here we show that translation also initiates at an internal start codon that enables independent expression of a separate reductase subunit, PgaMred. This confirms that the gene exhibits a rare viral-like arrangement of two overlapping reading frames that allows simultaneous expression of two alternative forms of the protein. Together, these two proteins associate to form a stable non-covalent complex, the native form of PgaM. The reductase subunit PgaMred is shown to provide enzyme stability and to affect the redox state of the oxygenase domain FAD. Finally, a model for the quaternary structure of the complex that explains the necessity for a nested gene system and the unusual behaviour of the protein subunits in vitro is presented.
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PMID:A nested gene in Streptomyces bacteria encodes a protein involved in quaternary complex formation. 1807 2

The key three-dimensional features of flavohemoglobins have been unveiled by X-ray crystallographic investigations carried out on the Alcaligenes eutrophus and Escherichia coli proteins. Flavohemoglobins are made of a globin domain fused with a ferredoxin reductase-like FAD binding module and display highly conserved sequences in the active sites of both the heme-binding domain and the flavin-binding domain. Structural studies are discussed and methodological approaches to the solution of the crystal structures and to the analysis of the relevant stereochemical properties of the active sites are presented. The understanding of the structural properties of flavohemoglobins serves as a guide for testing biological hypotheses and allows for a rational evaluation of structure-based alignments within the flavohemoglobin family.
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PMID:Structural studies on flavohemoglobins. 1823 33

Flavohemoglobins (flavoHbs) are made of a globin domain fused with a ferredoxin reductaselike FAD- and NAD-binding modules. These proteins are widely represented among bacteria and yeasts and represent a most challenging research subject in view of their high reactivity both as reductases and as oxidases. The functional annotations of flavoHbs are still controversial, and different physiological roles that are linked to cell responses to oxidative and/or nitrosative stress have been proposed. The flavoHb from Escherichia coli (HMP) has been the object of a large number of investigations to unveil its physiological role in the framework of bacterial resistance to nitrosative stress. HMP expression has been demonstrated to respond to the presence of NO in the culture medium, and an explicit mechanism has been proposed that involves NO scavenging and its reduction to N(2)O under anaerobic conditions. In contrast to (or together with) the anaerobic NO-reductase activity, HMP has also been shown to be able to catalyze the oxidation of NO to NO(3) (-) (NO-dioxygenase activity) both in vivo and in vitro in the presence of O(2) and NADH. HMP has also been shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. The alkylhydroperoxide reductase activity taken together with the unique lipid-binding properties of HMP suggests that this flavoHb may be involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.
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PMID:Flavohemoglobin: structure and reactivity. 1837 89

FAD synthetases (EC 2.7.7.2) catalyze biosynthesis of FAD from FMN and ATP. Monofunctional FAD synthetases are known to exist in mammals and yeast; bifunctional enzymes also catalyzing phosphorylation of riboflavin to FMN are known to exist in bacteria. Previously known eukaryotic enzymes with FAD synthetase activity have no sequence similarity to prokaryotic enzymes with riboflavin kinase and FAD synthetase activities. Proteins homologous to bacterial bifunctional FAD synthetases, yet shorter and lacking amino acid motifs at the C terminus, were found by bioinformatic analyses in vascular plant genomes, suggesting that plants contain a type of FAD synthetase previously known to exist only in prokaryotes. The Arabidopsis thaliana genome encodes two of such proteins. Both proteins, which we named AtRibF1 and AtRibF2, carry N-terminal extensions with characteristics of organellar targeting peptides. AtRibF1 and AtRibF2 cDNAs were cloned by reverse transcription-PCR. Only FAD synthetase activity was detected in the recombinant enzymes produced in Escherichia coli. FMN and ATP inhibited both enzymes. Kinetic parameters of AtRibF1 and AtRibF2 for the two substrates were similar. Confocal microscopy of protoplasts transformed with enhanced green fluorescence protein-fused proteins showed that AtRibF1 and AtRibF2 are targeted to plastids. In agreement with subcellular localization to plastids, Percoll-isolated chloroplasts from pea (Pisum sativum) synthesized FAD from imported riboflavin. Riboflavin kinase, FMN hydrolase, and FAD pyrophosphatase activities were detected in Percoll-isolated chloroplasts and mitochondria from pea. We propose from these new findings a model for subcellular distribution of enzymes that synthesize and hydrolyze flavin nucleotides in plants.
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PMID:Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize fad in plastids. 1871 32

In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give Delta G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. Delta G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of Delta G810 is insensitive to calmodulin binding. In addition, calmodulin binding to Delta G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in Delta G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.
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PMID:Exploring the electron transfer properties of neuronal nitric-oxide synthase by reversal of the FMN redox potential. 1885 62

The E1-like superfamily is central to ubiquitin (Ub) conjugation, biosynthesis of cysteine, thiamine, and MoCo, and several secondary metabolites. Yet, its functional diversity and evolutionary history is not well understood. We develop a natural classification of this superfamily and use it to decipher the major adaptive trends occurring in the evolution of the E1-like superfamily. Within the Rossmann fold, E1-like proteins are closest to NAD(P)/FAD-dependent dehydrogenases and S-AdoMet-dependent methyltransferases. Hence, their phosphotransfer activity is an independent catalytic "invention" with respect to such activities seen in other Rossmannoid folds. Sequence and structure analysis reveals a striking diversity of residues and structures involved in adenylation, sulfotransfer, and substrate binding between different E1-like families, allowing us to predict previously uncharacterized functional adaptations. E1-like proteins are fused to several previously undetected domains, such as a predicted sulfur transfer domain containing a novel superfamily of the TATA-binding protein fold, different types of catalytic domains, a novel winged helix-turn-helix domain and potential adaptor domains related to Ub conjugation. On the basis of these fusions, we develop a generalized model for the linking of E1 catalyzed adenylation/thiolation with further downstream reactions. This is likely to involve a dynamic interplay between the E1 active sites and diverse fused C-terminal domains. We also predict participation of E1-like domains in previously uncharacterized bacterial secondary metabolism pathways, new cysteine biosynthesis systems, such as those associated with archaeal O-phosphoseryl tRNA, metal-sulfur cluster assembly (e.g., in nitrogen fixation) and Ub-conjugation. Evolutionary reconstructions suggest that the last universal common ancestor contained a single E1-like domain possessing both phosphotransfer and thiolating activities and participating in multiple sulfotransfer reactions. The E1-like superfamily subsequently expanded to include 26 families clustering into three major radiations. These are broadly involved in Ub activation, cofactor and cysteine biosynthesis, and biosynthesis of secondary metabolites. In light of this, we present evidence that in eukaryotes other E1-like enzymes such as Urm1 were independently recruited for Ubl conjugation, probably functioning without conventional E2-like enzymes.
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PMID:Natural history of the E1-like superfamily: implication for adenylation, sulfur transfer, and ubiquitin conjugation. 1908 47

We described here the construction of two novel Saccharomyces cerevisiae strains in which the regulatory region of the SDH1 gene, coding for the succinate dehydrogenase flavoprotein subunit, was fused in frame to the reporter gene lacZ of E. coli, coding for beta-galactosidase. By this approach, SDH1 expression was studied in the yeast strain, flx1 delta-lacZ, lacking of a functional mitochondrial FAD translocator, Flx1p. The experiments described here are in line with the hypotesys that a correlation exists between defects in flavin cofactor homeostasis and mitochondrial apo-flavoprotein expression.
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PMID:Expression of succinate dehydrogenase flavoprotein subunit in saccharomyces cerevisiae studied by lacZ reporter strategy. Effect of FLX1 deletion. 1919 35

D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.
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PMID:Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase. 1926 68


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