Publications

• Flavin reductase P: structure of a dimeric enzyme that reduces flavin.

  Tanner J.J., Lei B., Tu S.-C., Krause K.L.

  We report the structure of an NADPH:FMN oxidoreductase (flavin reductase P) that is involved in bioluminescence by providing reduced FMN to luciferase. The 1.8 A crystal structure of flavin reductase P from Vibrio harveyi was solved by multiple isomorphous replacement and reveals that the enzyme i ... >> More

  We report the structure of an NADPH:FMN oxidoreductase (flavin reductase P) that is involved in bioluminescence by providing reduced FMN to luciferase. The 1.8 A crystal structure of flavin reductase P from Vibrio harveyi was solved by multiple isomorphous replacement and reveals that the enzyme is a unique dimer of interlocking subunits, with 9352 A2 of surface area buried in the dimer interface. Each subunit comprises two domains. The first domain consists of a four-stranded antiparallel beta-sheet flanked by helices on either side. The second domain reaches out from one subunit and embraces the other subunit and is responsible for interlocking the two subunits. Our structure explains why flavin reductase P is specific for FMN as cofactor. FMN is recognized and tightly bound by a network of 16 hydrogen bonds, while steric considerations prevent the binding of FAD. A flexible loop containing a Lys and an Arg could account for the NADPH specificity. The structure reveals information about several aspects of the catalytic mechanism. For example, we show that the first step in catalysis, which is hydride transfer from C4 of NADPH to cofactor FMN, involves addition to the re face of the FMN, probably at the N5 position. The limited accessibility of the FMN binding pocket and the extensive FMN-protein hydrogen bond network are consistent with the observed ping-pong bisubstrate--biproduct reaction kinetics. Finally, we propose a model for how flavin reductase P might shuttle electrons between NADPH and luciferase. << Less

  Biochemistry 35:13531-13539(1996) [PubMed] [EuropePMC]

• Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

  Jablonski E., DeLuca M.

  Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kine ... >> More

  Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase. << Less

  Biochemistry 17:672-678(1978) [PubMed] [EuropePMC]

• Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli.

  Eichhorn E., van der Ploeg J.R., Leisinger T.

  The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal ... >> More

  The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of M(r) 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin. The SsuD protein was purified to >98% purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH(2) and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mM Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M(r) 181,000. These results demonstrate that SsuD is a broad range FMNH(2)-dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes. SsuE is the FMN reducing enzyme providing SsuD with FMNH(2). << Less

  J. Biol. Chem. 274:26639-26646(1999) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• 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.

• Purification and characterization of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase.

  Zenno S., Kobori T., Tanokura M., Saigo K.

  ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to ... >> More

  ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome. << Less

  Biosci. Biotechnol. Biochem. 62:1978-1987(1998) [PubMed] [EuropePMC]

• Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi.

  Jablonski E., DeLuca M.

  The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a cru ... >> More

  The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is chromatography on a 5'-AMP-Sepharose 4B affinity column which results in approximately a 50-fold purification to a final specific activity of 31 mumol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase has been purified to a final specific activity of 51 mumol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. Molecular weights of 30 000 and 40 000 and Km's of 4.75 X 10(-5) M NADH and 4.0 X 10(-5) M NADPH have been determined for the pure NADH and NADPH specific FMN reductases, respectively. The NADPH specific FMN reductase does not utilize NADH, while the NADH specific enzyme does dehydrogenate NADPH with a maximal velocity one-tenth of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated. << Less

  Biochemistry 16:2932-2936(1977) [PubMed] [EuropePMC]

• Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme.

  Lei B., Liu M., Huang S., Tu S.-C.

  NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N ... >> More

  NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism. << Less

  J. Bacteriol. 176:3552-3558(1994) [PubMed] [EuropePMC]

• 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.

• Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase.

  Lei B., Tu S.C.

  The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to ... >> More

  The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NADPH, exhibited a ping-pong kinetic pattern with a Km, FMN of 8 microM and a Km,NADPH of 20 microM in a single-enzyme spectrophotometric assay monitoring the NADPH oxidation. However, the kinetic mechanism of FRP was changed to a sequential pattern with a Km,FMN of 0.3 microM and a Km,NADPH of 0.02 microM in a luciferase-coupled assay measuring light emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxidoreductase FRG showed the same ping-pong mechanism in both the single-enzyme spectrophotometric and the luciferase-coupled assays. Moreover, for the FRP, FMN at concentrations over 2 microM significantly inhibited the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation was detected under identical conditions. These results are consistent with a scheme that the reduced flavin cofactor of FRP is preferentially utilized by luciferase for light emission, the reduced flavin product generated by the reductase is primarily channeled into a dark oxidation, and luciferase competes against flavin substrate in reacting with the FRP reduced flavin cofactor. An FRP derivative containing 2-thioFMN as the cofactor was also used to further examine the mechanism of flavin transfer. Results again indicate a preferential utilization of the reductase reduced flavin cofactor by luciferase for the bioluminescence reaction. << Less

  Biochemistry 37:14623-14629(1998) [PubMed] [EuropePMC]

• Vibrio harveyi NADPH:FMN oxidoreductase: preparation and characterization of the apoenzyme and monomer-dimer equilibrium.

  Liu M., Lei B., Ding Q., Lee J.C., Tu S.C.

  A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with > or =80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 microM at 23 degrees C. The reconstituted holoenzyme was catalytically a ... >> More

  A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with > or =80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 microM at 23 degrees C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme. 2-ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2-thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4 degrees C by equilibrium ultracentrifugation to undergo dimerization with dissociation constants of 1.8 and 3.3 microM, respectively. << Less

  Arch Biochem Biophys 337:89-95(1997) [PubMed] [EuropePMC]