Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An L-amino acid oxidase (LAO), designated as TJ-LAO, was purified to homogeneity from the venom of Trimeresurus jerdonii by Sephadex G-100 and Q Sepharose HP chromatography. The molecular weight of this enzyme was 110 kD as estimated by analytical gel filtration and was 55 kD by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two subunits. The enzyme has an absorption spectrum characteristic of flavoproteins, containing 2 moles of FMN per mole of enzyme. The N-terminal sequence of TJ-LAO shares high homology with other viperid snake venom LAOs. Homology with elapid venom LAO is lower. TJ-LAO inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus megaterium. The antibacterial effect associated with LAO activity was elminated with the addition of catalase. Platelets in platelet-rich plasma aggregated upon the addition of TJ-LAO. The enzyme-induced aggregation was inhibited by catalase, suggesting formation of H2O2 was essential for TJ-LAO to induce platelet aggregation. These results showed H2O2 formation is important for the biological effects of LAO.
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PMID:L-amino acid oxidase from Trimeresurus jerdonii snake venom: purification, characterization, platelet aggregation-inducing and antibacterial effects. 1250 78

Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E. faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent Km values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent Vmax was 86.2 microM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria.
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PMID:Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis. 1500 65

Riboflavin sensitized photodynamic modifications of high molecular weight Kininogen (HMWK) isolated from sheep (Avis-arias) plasma leads to inactivation of antiproteinase activity and formation of aggregated products. A continued disappearance of the inhibitory activity towards papain and formation of high molecular weight adducts was observed with increasing concentration of riboflavin and varying time periods of incubation reaching a maximum value of over 85% (loss in activity). Aggregates resisted dissociation upon heating at 100 degrees C in 1% SDS. Aggregation and photoinactivation of HMWK was promoted by the substitution of H2O for deuterium oxide (D2O), which is known to prolong the life span of singlet oxygen, and suppressed by sodium azide a known singlet oxygen quencher. Mannitol and thiourea (hydroxyl radical scavenger) did not protect the antiproteinase activity of HMWK. Treatment with reducing agent resulted in decrease of the aggregated products suggesting the possible involvement of disulfide linkages in protein crosslinking. Tryptophan fluorescence was completely lost and significant production of dityrosine was detected in photoinactivated HMWK aggregates. Changes in the far Ultra violet circular dichroism (u.v.c.d.) spectrum of HMWK was indicative of loss of secondary structure. Analysis of modifications induced in HMWK by riboflavin reveals that the processes proceed via a singlet oxygen mediated pathway. It is concluded that the susceptibility of HMWK to oxidation may arise from oxidative modifications by reactive oxygen species generated in plasma.
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PMID:Modification of sheep plasma kininogen by free radicals. 1519 Sep 36

Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30-40 degrees C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2',6,6'-tetranitro-4,4'-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.
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PMID:Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene. 1578 4

A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The molecular mass of the purified enzyme was determined to be 120 kDa by gel filtration and the subunit molecular mass was calculated to be 48 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicating a dimeric structure. The N-terminal amino acid sequence of the purified TdsA was determined to be MRQMHLAGFFAAGNTHH, which revealed no significant similarity to any other known amino acid sequences. The purified TdsA absolutely required an oxidoreductase for its activity. This oxidoreductase (TdsD) was also purified to homogeneity, and its molecular size was calculated to be 50 kDa and 25 kDa by gel filtration and SDS-PAGE, respectively. TdsD was completely FMN-dependent, and FAD could not act as a cofactor. The N-terminal amino acid sequence of the purified TdsD was determined to be TSQTAEQSIAPIVAQYRHPEQPISALFVNR, which showed significant similarity to kinesin-like protein (44% identity). The optimal temperatures for the activity of TdsA and TdsD were 45 degrees C and 55 degrees C, respectively. Both enzymes showed optimal activity at pH 5.5. TdsA was slightly inhibited by sulfate, but not by 2-hydroxybiphenyl (2-HBP), which is another end product of DBT. TdsA showed higher activity toward bulkier substrates than its mesophilic counterpart, DszA. These properties suggest the applicability of biodesulfurization to the processing of actual petroleum fractions.
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PMID:Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11-2. 1623 19

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.
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PMID:Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase. 1664 57

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.
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PMID:Molecular cloning and characterization of a large subunit of Salmonella typhimurium glutamate synthase (GOGAT) gene in Escherichia coli. 1682 Jul 60

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.
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PMID:Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase. 1704 78

Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.
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PMID:Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae. 1883 35

FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent K(m) and V(max) values for FMN equal to 1.3+/-0.7 microM and 4.4+/-1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 microM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.
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PMID:Mitochondrial localization of human FAD synthetase isoform 1. 2006 May 5


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