Publications

• Cloning and nucleotide sequence of the gene for NADH:FMN oxidoreductase from Vibrio harveyi.

  Izumoto Y., Mori T., Yamamoto T.

  The gene encoding the enzyme NADH:FMN oxidoreductase (EC 1.6.99.3) from Vibrio harveyi has been isolated from a recombinant library of genomic DNA and sequenced. The deduced amino acid sequence, 237 amino acids long, shows 48% identity with E. coli NAD(P)H:flavin oxidoreductase and 40% identity wi ... >> More

  The gene encoding the enzyme NADH:FMN oxidoreductase (EC 1.6.99.3) from Vibrio harveyi has been isolated from a recombinant library of genomic DNA and sequenced. The deduced amino acid sequence, 237 amino acids long, shows 48% identity with E. coli NAD(P)H:flavin oxidoreductase and 40% identity with Vibrio harveyi luxG gene product. << Less

  Biochim. Biophys. Acta 1185:243-246(1994) [PubMed] [EuropePMC]

• Flavin mononucleotide reductase of luminous bacteria.

  Duane W., Hastings J.W.

  NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of lumino ... >> More

  NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex. << Less

  Mol Cell Biochem 6:53-64(1975) [PubMed] [EuropePMC]

• Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

  Xu Y., Mortimer M.W., Fisher T.S., Kahn M.L., Brockman F.J., Xun L.

  Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygen ... >> More

  Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation. << Less

  J. Bacteriol. 179:1112-1116(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase.

  Watanabe H., Hastings J.W.

  Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited ... >> More

  Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4 degrees but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase. << Less

  Mol. Cell. Biochem. 44:181-187(1982) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harveyi.

  Gerlo E., Charlier J.

  Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient ... >> More

  Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient than the NADPH-specific one with respect to the bioluminescent reaction. << Less

  Eur J Biochem 57:461-467(1975) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure and function of YcnD from Bacillus subtilis, a flavin-containing oxidoreductase.

  Morokutti A., Lyskowski A., Sollner S., Pointner E., Fitzpatrick T.B., Kratky C., Gruber K., Macheroux P.

  YcnD from the gram-positive bacterium Bacillus subtilis is a member of a family of bacterial proteins that act as NADH-and/or NADPH-dependent oxidoreductases. Here, we report for the first time on the biochemical characterization of the purified protein, demonstrating that YcnD is an FMN-containin ... >> More

  YcnD from the gram-positive bacterium Bacillus subtilis is a member of a family of bacterial proteins that act as NADH-and/or NADPH-dependent oxidoreductases. Here, we report for the first time on the biochemical characterization of the purified protein, demonstrating that YcnD is an FMN-containing enzyme that can be reduced by NADH or NADPH (Km = 6.4 and 4.4 microM, respectively). In the presence of free FMN as the electron-accepting substrate, the latter reductant showed a ping-pong Bi-Bi reaction mechanism, whereas utilization of NADH is competitively inhibited by this substrate. This finding suggests that NADPH is the physiological reductant of the enzyme. We also show that YcnD reduces nitro-organic compounds, chromate, and a series of azo dyes. The reduction of azo dyes appears to be mediated by free reduced FMN because the reaction is considerably slower in its absence. Structure determination by X-ray crystallography revealed that YcnD folds into a three layer alpha-beta-alpha sandwich strongly resembling the topology of the NADH oxidase superfamily. Similar to homologous bacterial oxidoreductase, YcnD forms homodimers with an extended dimer interface. The biochemical data and the structure are discussed in light of the putative physiological function of YcnD as an oxidoreductase delivering reduced FMN to enzymes that require the reduced cofactor for activity. << Less

  Biochemistry 44:13724-13733(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from 'Chelatobacter' strain ATCC 29600.

  Uetz T., Schneider R., Snozzi M., Egli T.

  An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily d ... >> More

  An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H2O2. Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg(2+)-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 microM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo. << Less

  J. Bacteriol. 174:1179-1188(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Catalysis of a flavoenzyme-mediated amide hydrolysis.

  Mukherjee T., Zhang Y., Abdelwahed S., Ealick S.E., Begley T.P.

  A new pyrimidine catabolic pathway (the Rut pathway) was recently discovered in Escherichia coli K12. In this pathway, uracil is converted to 3-hydroxypropionate, ammonia, and carbon dioxide. The seven-gene Rut operon is required for this conversion. Here we demonstrate that the flavoenzyme RutA c ... >> More

  A new pyrimidine catabolic pathway (the Rut pathway) was recently discovered in Escherichia coli K12. In this pathway, uracil is converted to 3-hydroxypropionate, ammonia, and carbon dioxide. The seven-gene Rut operon is required for this conversion. Here we demonstrate that the flavoenzyme RutA catalyzes the initial uracil ring-opening reaction to give 3-ureidoacrylate. This reaction, while formally a hydrolysis reaction, proceeds by an oxidative mechanism initiated by the addition of a flavin hydroperoxide to the C4 carbonyl. While peroxide-catalyzed amide hydrolysis has chemical precedent, we are not aware of a prior example of analogous chemistry catalyzed by flavin hydroperoxides. This study further illustrates the extraordinary catalytic versatility of the flavin cofactor. << Less

  J. Am. Chem. Soc. 132:5550-5551(2010) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.