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 DAO1 gene of Trigonopsis variabilis encoding a D-amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution. The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D-amino acid oxidases. The open reading frame of the T. variabilis DAO1 gene was interrupted by an intron. The Dao1p sequence displays two regions, one in the N-terminal section--the FAD binding site--and the other near the C-terminal region that contains conserved signatures found in all the D-amino acid oxidases. The three C-terminal amino acids suggest that the enzyme may be located in peroxisomes. Northern blot experiments showed that no transcriptional activation occurred in the presence of D-methionine. The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter. In K. lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild-type strain of T. variabilis. The yeast expression system we describe may constitute an alternative source for the production of D-amino acid oxidases at industrial level.
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PMID:Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis. 943 46

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.
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PMID:Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. 950 Dec 15

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. CDH contains one heme b and one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochrome c. In this report, a thermostable CDH from the thermophilic ascomycete Sporotrichum thermophile has been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60 degrees C, and the activation energy for the reaction was 26.3 kJ/mol. The Km and kcat were temperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library of S. thermophile with a polyclonal antisera raised against Phanerochaete chrysosporium CDH. The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da. S. thermophile CDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases from Trichoderma reesei. Comparison with the CDH sequences of P. chrysosporium and Trametes versicolor identified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs. With regard to the amino acid composition, S. thermophile CDH has more disulfide linkages and acidic and basic amino acids compared to CDHs from P. chrysosporium and T. versicolor.
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PMID:Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile. 1032 16

Nitric oxide synthases (NOSs) are ubiquitous in living organisms. However, little is known about the evolution of this large gene family. The first inducible NOS to be described from an invertebrate regulates malaria parasite (Plasmodium spp.) development in the mosquito Anopheles stephensi. This single copy gene shows the highest homology to the vertebrate neuronal isoforms, followed by decreasing homology to endothelial and inducible isoforms. The open reading frame of 1247 amino acids is encoded by 19 exons, which span approximately 33 kilobases. More than 50% of the mosquito exons, distributed around the putative heme, calmodulin, and FAD/NADPH cofactor-binding domains, are conserved with those of the three human genes. Repetitive elements identified within the larger introns include a polymorphic dinucleotide repeat, two tandem repeats, and a putative miniature inverted repeat transposable element. Sequence analysis and primer extension indicate that the upstream promoter is 'TATA-less' with multiple transcription start sites within approximately 250 base pairs of the initiation methionine. Transcription factor binding sites in the 5'-flanking sequence demonstrate a bipartite distribution of lipopolysaccharide- and inflammatory cytokine-responsive elements that is strikingly similar to that described for vertebrate inducible NOS gene promoters.
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PMID:Gene structure and polymorphism of an invertebrate nitric oxide synthase gene. 1033 18

Methionine synthase reductase (MSR) deficiency is an autosomal recessive disorder of folate/cobalamin metabolism leading to hyperhomocysteinemia, hypo- methioninemia and megaloblastic anemia. Deficiency in MSR activity occurs as the result of a defect in the MSR enzyme, which is required for the reductive activation of methionine synthase (MS). MS itself is responsible for the folate/cobalamin-dependent conversion of homo- cysteine to methionine. We have recently cloned the cDNA corresponding to the MSR protein, a novel member of the ferredoxin-NADP(+)reductase (FNR) family of electron transferases. We have used RT-PCR, heteroduplex, single-strand conformation poly- morphism (SSCP) and DNA sequence analyses to reveal 11 mutations in eight patients from seven families belonging to the cblE complementation group of patients of cobalamin metabolism that is defective in the MSR protein. The mutations include splicing defects leading to large insertions or deletions, as well as a number of smaller deletions and point mutations. Apart from an intronic substitution found in two unrelated patients, the mutations appear singular among individuals. Of the eleven, three are nonsense mutations, allowing for the identification of two patients for whom little if any MSR protein should be produced. The remaining eight involve point mutations or in-frame disruptions of the coding sequence and are distributed throughout the coding region, including proposed FMN, FAD and NADPH binding sites. These data demonstrate a unique requirement for MSR in the reductive activation of MS.
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PMID:Molecular basis for methionine synthase reductase deficiency in patients belonging to the cblE complementation group of disorders in folate/cobalamin metabolism. 1048 69

The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned and the nucleotide sequence of the enzyme gene was clarified. An open reading frame of 1,413 bp starting at an ATG methionine codon was found, which encodes a protein of 471 amino acid residues, the calculated molecular weight of which is 51,518. The amino acid sequence of P. vulgaris was 58.6% identical with the L-amino acid deaminase of P. mirabilis. A significantly conserved sequence was found around the FAD-binding sequence of flavo-proteins. The partially purified wild and recombinant enzymes had the same substrate specificity for L-amino acids to form the respective keto-acids, however not for D-amino acids.
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PMID:Cloning of L-amino acid deaminase gene from Proteus vulgaris. 1066 62

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.
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PMID:Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia. 1073 51

The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of </=4.5. NADH titration of the mutant peroxidase yields the same EH(2) intermediate, but in contrast to the behavior of wild-type enzyme, this species can be reduced directly to an EH(4).NAD(+) complex. Kinetic analyses demonstrate that the R303M mutant is severely compromised, although active, with k(cat) = 3 s(-)(1) at pH 7.0, 5 degrees C; enzyme-monitored turnover results indicate that the steady-state consists predominantly of an E-FADH(2).NAD(+) species. When the oxidized mutant is reacted anaerobically with 0.9 equiv of NADH/FAD, a clearly biphasic pattern is observed at 450 nm; relatively rapid flavin reduction is followed by reoxidation at 2.6-2.7 s(-)(1) ( approximately k(cat)). Thus replacement of Arg303 with Met leads to an altered peroxidase form in which the rate-limiting step in turnover is the intramolecular transfer of electrons from FADH(2) --> Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.
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PMID:Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure. 1095 25

Pantothenic acid and beta-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for beta-alanine and pantothenic acid biosynthesis. Overexpression of FMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required beta-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 and YIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in beta-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for beta-alanine biosynthesis; spe1Delta, spe2Delta, spe3Delta, and spe4Delta are all beta-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the beta-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms.
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PMID:Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine. 1115 94

D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host. The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor. The enzyme was active toward several D-amino acids such as D-Ala, D-Met, and D-Ser. An alcohol oxidase-depleted strain (aod1delta) was found to be a more suitable host for DAO production than the wild-type strain. Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1delta strain. Finally, an aod1delta strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30% of the total soluble proteins.
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PMID:Characterization and high-level production of D-amino acid oxidase in Candida boidinii. 1133 Jun 78


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