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Crystallization and Preliminary Crystallographic Analysis of the Major NAD(P)H:FMN Oxidoreductase ofVibrio fischeriATCC 7744
JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. 0070
117, 70–72 (1996)
CRYSTALLIZATION NOTE Crystallization and Preliminary Crystallographic Analysis of the Major NAD(P)H:FMN Oxidoreductase of Vibrio fischeri ATCC 7744 HIDEAKI KOIKE, HIROSHI SASAKI,
AND
MASARU TANOKURA1
Biotechnology Research Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan AND
SHUHEI ZENNO2
AND
KAORU SAIGO
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received November 2, 1995, and in revised form January 16, 1996
coupled reaction The major flavin reductase from Vibrio fischeri, FRase I, has been crystallized in the presence of FMN by the vapor diffusion method using polyethylene glycol 4000 as a precipitant. The crystals belonged to the monoclinic space group C2 with unit cell dimensions, a 5 101.6 Å, b 5 63.2 Å, c 5 74.4 Å, and b 5 100.0°. The crystals are expected to contain two FRase I molecules per asymmetric unit. The crystals diffracted X-rays to at least 2.2 Å resolution and are appropriate for structural analysis at high resolution. r 1996 Academic Press, Inc.
FMN 1 NAD(P)H 1 H1 = FMNH2 1 NAD(P)1 FMNH2 1 R-CHO 1 O2 = FMN 1 R-COOH 1 H2O 1 light. Many flavin reductases have been enzymatically identified in various organisms ranging from bacteria to humans (Puget and Michelson, 1972; Duane and Hastings, 1975; Yubisui et al., 1977; Hasan and Nester, 1978; Lo and Reeves, 1980; Fontecave et al., 1987), including luminous bacteria such as Vibrio fischeri (Hastings et al., 1965; Gunsalus-Miguel et al., 1972; Duane and Hastings, 1975; Tu et al., 1979; Zenno and Saigo, 1994; Zenno et al., 1994), Vibrio harveyi (Puget and Michelson, 1972; Gerlo and Charlier, 1975; Jablonski and DeLuca, 1977; Michaliszyn et al., 1977; Lavi et al., 1990; Zenno and Saigo, 1994), Photobacterium phosphoreum (Puget and Michelson, 1972; Lavi et al., 1990), Photobacterium leiognathi (Puget and Michelson, 1972), and Photohabdus luminescences (Schmidt et al., 1989; Zenno and Saigo, 1994). Their molecular weights, substrate specificities, and reaction mechanisms vary considerably from enzyme to enzyme (Duane and Hastings, 1975; Jablonski and DeLuca, 1978, Tu et al., 1979; Nefsky and DeLuca, 1982; Watanabe and Hastings, 1982; Hastings et al., 1985), suggesting that flavin
NAD(P)H:flavin oxidoreductase (EC 1.6.8) (also called flavin reductase) catalyzes the reduction of flavins, including riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), at the expense of reduced pyridine nucleotides (NAD(P)H). In luminous bacteria, this enzyme plays an important role in supplying reduced FMN (FMNH2 ) to the luminescence reaction (Hastings et al., 1985; Meighen, 1991). The luminescence reaction involves a heterodimeric enzyme, luciferase, which catalyzes the oxidation of FMNH2 and long-chain aliphatic aldehyde (R-CHO) by molecular oxygen with the emission of a blue–green light according to the 1 To whom correspondence and reprint requests should be addressed. 2 Present address: Yokohama Research Center, Chisso Corporation, 5-1 Ookawa, Kanazawa-ku, Yokohama 236, Japan.
1047-8477/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
70
CRYSTALLIZATION NOTE
reductases are not necessarily related to one another in sequence or evolution. So far, a few studies have been reported about the reaction mechanism of the major flavin reductase of V. fischeri. Tu et al. partially purified the enzyme from V. fischeri which had no flavin and analyzed the reaction mechanism by steady-state kinetics (Tu et al., 1979). The mechanism of the enzyme catalysis was the ping-pong bisubstrate-biproduct mechanism. The enzyme was inactivated by N-ethylmaleimide in the presence of NADH, but was not inactivated alone or in the presence of FMN. Tu et al. concluded that the flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis. We have cloned the gene encoding the major NAD(P)H-flavin oxidoreductase from V. fischeri ATCC 7744 and expressed in Escherichia coli (Zenno et al., 1994). The enzyme expressed in E. coli binds FMN and is colored yellow (Inouye, 1994). Biochemical studies suggest that only bound FMN is responsible for oxidoreduction (Koike et al., unpublished data). The detailed catalytic mechanism has not been revealed. In order to gain an insight into the details of the catalytic mechanism and the substrate specificity of the enzyme, a high-resolution three-dimensional structure has to be elucidated for the enzyme. Here we report crystallization and preliminary X-ray crystallographic analysis of the major flavin reductase of V. fischeri ATCC 7744. E. coli D1210 cells (de Boer et al., 1983), carrying an FRase I expression plasmid pFR7 (Zenno et al., 1994), were cultured in LB media containing 200 µg/ml ampicillin at 37°C for 7 hr. Then cells were harvested by centrifugation at 10 000g for 30 min, and FRase I was purified as described previously (Zenno et al., 1994). The protein was purified further by loading onto a phenyl-Sepharose column (Pharma-
FIG. 1.
Plate-like yellow crystals of FRase I in a sitting drop.
71
FIG. 2. Precession photographs taken with a precession angle of 20° for 24 hr. (a) hk0 plane; systematic absences on h 1 k were observed. (b) h0l plane; systematic absences along h00 line were observed and an angle between a* and c* was 80°.
cia) in 50 mM Tris-HCl (pH 7.0) containing 20% (w/v) ammonium sulfate and subsequently eluting by a linear gradient against the same buffer without ammonium sulfate. The pure FRase I fraction was dialyzed against 5 mM Tris-HCl (pH 7.0) and concentrated to approximately 50 mg/ml using a Centricon-10 (Amicon). Crystallization was accomplished at 25°C as follows. Crystals were grown using the sitting drop vapor diffusion method by mixing equal volumes of the protein solution with a reservoir solution that contained 30% (w/v) polyethylene glycol 4000, 0.1 mM FMN, 0.2 M sodium acetate in 0.1 M Tris-HCl (pH 8.5). Plate-like yellow crystals (0.2 3 0.2 3 0.1 mm3 ) appeared over 1 month (Fig. 1). Preliminary X-ray diffraction experiments were performed using a precession camera mounted on a 4057V1 sealed tube machine (Rigaku) and an Rigaku R-AXIS IIc diffractometer equipped with imaging plates coupled to an RU-200R rotating anode generator. The sealed tube machine was operated at 40 kV and 30 mA, and a 0.5-mm collimator was used. The rotating anode was operated at 50 kV and 100 mA and focused by a double focusing mirror. To collect and process the R-AXIS data, the standard Rigaku R-AXIS software was used. Precession photographs and the R-AXIS data revealed that the crystals of FRase I belonged to the monoclinic space group C2 with unit cell dimensions, a 5 101.6 Å, b 5 63.2 Å, c 5 74.4 Å, and b 5 100.0° (Fig. 2). The assumption of one subunit per asymmetric unit leads to a Vm (crystal volume per unit of protein mass) of 4.72 Å3/Da, corresponding to a solvent content of 74% (Matthews, 1968). If two subunits exist in the asymmetric unit, a Vm value is 2.35 Å3/Da (solvent content of 48%). Because the enzyme may act as a dimer (Zenno et al., unpublished data), we expect two molecules per asymmetric unit. Intensity data were collected by using a data
72
CRYSTALLIZATION NOTE
TABLE I Statistics of the Collected X-Ray Diffraction Intensity Data of V. fischeri FRase I Resolution (Å)
Completion (%)
Intensity
Sigma
Merging R-factor
–7.14 7.14–5.05 5.05–4.13 4.13–3.58 3.58–3.20 3.20–2.92 2.92–2.70 2.70–2.53 2.53–2.38 2.38–2.26 2.26–2.16
72.2 96.3 92.9 98.2 100.0 99.1 97.7 97.1 92.6 89.8 84.1
597 675 1050 979 763 516 340 255 212 180 143
126.9 136.5 193.6 154.6 99.7 73.7 51.3 37.2 33.7 31.6 29.1
0.073 0.071 0.072 0.074 0.073 0.079 0.086 0.091 0.098 0.110 0.127
collection system at the BL6A station in the Photon Factory at the National Laboratory for High Energy Physics, by combining the Weissenberg camera for macromolecular crystallography, an imaging plate, a Fuji image reader BA100, and the data reduction program WEIS with a synchrotron radiation (Sakabe, 1983, 1991; Higashi, 1989). The crystals diffracted X-rays to at least 2.2 Å resolution and are good for a high-resolution structure determination. The data statistics are shown in Table I. In order to determine the three-dimensional structure of FRase I, the preparation of the crystals of heavyatom derivatives of the enzyme is in progress. We thank Professor N. Sakabe (Tsukuba University) for his encouragement and interest during the experiment. We also thank Dr. M. E. P. Murphy for reading the manuscript and for advice. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan. REFERENCES de Boer, H. A., Comstock, L. J., and Vasser, M. (1983) The tac promoter: A functional hybrid derived from the trp and lac promoter, Proc. Natl. Acad. Sci. USA 80, 21–25. Duane, W., and Hastings, J. W. (1975) Flavin mononucleotide reductase of luminous bacteria, Mol. Cell. Biochem. 6, 53–64. Fontecave, M., Eliasson, R., and Reichard, P. (1987) NAD(P)H: flavin oxidoreductase of Escherichia coli: A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase, J. Biol. Chem. 262, 12325–12331. Gerlo, E., and Charlier, J. (1975) Identification of NADH-specific and NADPH-specific FMN reductases in Benechea harveyi, Eur. J. Biochem. 57, 461–467. Gunsalus-Miguel, A., Meighen, E. A., Nicoli, M. Z., Nealson, K. H., and Hasting, J. W. (1972) Purification and properties of bacterial luciferase, J. Biol. Chem. 247, 398–404. Hasan, N., and Nester, E. W. (1978) Purification and characterization of NADPH-dependent flavin reductase: an enzyme required for the activation of chorismate synthase in Bacillus subtilis, J. Biol. Chem. 253, 4987–4992. Hastings, J. W., Potrikus, C. J., Gupta, S. C., Kurfurst, M., and Makemson, J. C. (1985) Biochemistry and physiology of bioluminescent bacteria, Adv. Microb. Physiol. 26, 235–291.
Hastings, J. W., Riley, W. H., and Massa, J. (1965) The purification, properties, and chemiluminescent quantum yield of bacterial luciferase, J. Biol. Chem. 240, 1473–1481. Higashi, T. (1989) The processing of diffraction data taken on a screenless Weissenberg camera for macromolecular crystallography, J. Appl. Crystallogr. 22, 9–18. Inouye, S. (1994) NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein, FEBS Lett. 347, 163–168. Jablonski, E., and DeLuca, M. (1977) Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckae harveyi, Biochemistry 16, 2932–2936. Lavi, J. T., Raunio, R. P., and Stahlberg, T. H. (1990) Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications, J. Biolumin. Chemilumin. 5, 187–192. Lo, H.-S., and Reeves, R. E. (1980) Purification and properties of NADPH:flavin oxidoreductase from Entamoeba histolytica, Mol. Biochem. Parasitol. 2, 23–30. Matthews, B. W. (1968) Solvent content of protein crystals, J. Mol. Biol. 33, 491–497. Meighen, E. A. (1991) Molecular biology of bacterial bioluminescence, Microbiol. Rev. 55, 123–142. Michaliszyn, G. A., Wing, S. S., and Meighen, E. A. (1977) Purification and properties of NAD(P)H:flavin oxidoreductase from the luminescence bacterium, Beneckea harveyi, J. Biol. Chem. 252, 7495–7499. Nefsky, B., and DeLuca, M. (1982) Studies on the NADH and NADPH:riboflavin 58-phosphate (FMN) oxidoreductases from Beneckea harveyi: Characterization of the FMN binding sites, Arch. Biochem. Biophys. 216, 10–16. Puget, K., and Michelson, A. M. (1972) Studies in bioluminescence. VII. Bacterial NADH:flavin mononucleotide oxidoreductase, Biochimie 54, 1197–1204. Sakabe, N. (1983) A focusing Weissenberg camera with multilayerline screens for macromolecular crystallography, J. Appl. Crystallogr. 16, 542–547. Sakabe, N. (1991) X-ray diffraction data collection system for modern protein crystallography with a Weissenberg camera and an imaging plate using synchrotron radiation, Nucl. Instrum. Methods A303, 448–463. Schmidt, T. M., Kopecky, K., and Nealson, K. H. (1989) Bioluminescence of insect pathogen Xenorhabdus luminescence, Appl. Envirn. Microbiol. 55, 2607–2612. Tu, S.-C., Becvar, J. E., and Hastings, J. W. (1979) Kinetic study on the mechanism of bacterial NAD(P)H:flavin oxidoreductase, Arch. Biochem. Biophys. 193, 110–116. Watanabe, H., and Hastings, J. W. (1982) Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase, Mol. Cell. Biochem. 44, 181–187. Yubisui, T., Matsuki, T., Tanishima, K., Takeshita, M., and Yoneyama, Y. (1977) NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme, Biochem. Biophys. Res. Commun. 76, 174–182. Zenno, S., and Saigo, K. (1994) Identification of the gene encoding NAD(P)H-Flavin oxidoreductase that are similar in sequence to Eschericia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harbeyi, and Vibrio orientalis, J. Bacteriol. 176, 3544–3551. Zenno, S., Saigo, K., Kanoh, H., and Inouye, S. (1994) Identification of the gene encoding the major NAD(P)H-Flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744, J. Bacteriol. 176, 3536–3543.
117, 70–72 (1996)
CRYSTALLIZATION NOTE Crystallization and Preliminary Crystallographic Analysis of the Major NAD(P)H:FMN Oxidoreductase of Vibrio fischeri ATCC 7744 HIDEAKI KOIKE, HIROSHI SASAKI,
AND
MASARU TANOKURA1
Biotechnology Research Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan AND
SHUHEI ZENNO2
AND
KAORU SAIGO
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received November 2, 1995, and in revised form January 16, 1996
coupled reaction The major flavin reductase from Vibrio fischeri, FRase I, has been crystallized in the presence of FMN by the vapor diffusion method using polyethylene glycol 4000 as a precipitant. The crystals belonged to the monoclinic space group C2 with unit cell dimensions, a 5 101.6 Å, b 5 63.2 Å, c 5 74.4 Å, and b 5 100.0°. The crystals are expected to contain two FRase I molecules per asymmetric unit. The crystals diffracted X-rays to at least 2.2 Å resolution and are appropriate for structural analysis at high resolution. r 1996 Academic Press, Inc.
FMN 1 NAD(P)H 1 H1 = FMNH2 1 NAD(P)1 FMNH2 1 R-CHO 1 O2 = FMN 1 R-COOH 1 H2O 1 light. Many flavin reductases have been enzymatically identified in various organisms ranging from bacteria to humans (Puget and Michelson, 1972; Duane and Hastings, 1975; Yubisui et al., 1977; Hasan and Nester, 1978; Lo and Reeves, 1980; Fontecave et al., 1987), including luminous bacteria such as Vibrio fischeri (Hastings et al., 1965; Gunsalus-Miguel et al., 1972; Duane and Hastings, 1975; Tu et al., 1979; Zenno and Saigo, 1994; Zenno et al., 1994), Vibrio harveyi (Puget and Michelson, 1972; Gerlo and Charlier, 1975; Jablonski and DeLuca, 1977; Michaliszyn et al., 1977; Lavi et al., 1990; Zenno and Saigo, 1994), Photobacterium phosphoreum (Puget and Michelson, 1972; Lavi et al., 1990), Photobacterium leiognathi (Puget and Michelson, 1972), and Photohabdus luminescences (Schmidt et al., 1989; Zenno and Saigo, 1994). Their molecular weights, substrate specificities, and reaction mechanisms vary considerably from enzyme to enzyme (Duane and Hastings, 1975; Jablonski and DeLuca, 1978, Tu et al., 1979; Nefsky and DeLuca, 1982; Watanabe and Hastings, 1982; Hastings et al., 1985), suggesting that flavin
NAD(P)H:flavin oxidoreductase (EC 1.6.8) (also called flavin reductase) catalyzes the reduction of flavins, including riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), at the expense of reduced pyridine nucleotides (NAD(P)H). In luminous bacteria, this enzyme plays an important role in supplying reduced FMN (FMNH2 ) to the luminescence reaction (Hastings et al., 1985; Meighen, 1991). The luminescence reaction involves a heterodimeric enzyme, luciferase, which catalyzes the oxidation of FMNH2 and long-chain aliphatic aldehyde (R-CHO) by molecular oxygen with the emission of a blue–green light according to the 1 To whom correspondence and reprint requests should be addressed. 2 Present address: Yokohama Research Center, Chisso Corporation, 5-1 Ookawa, Kanazawa-ku, Yokohama 236, Japan.
1047-8477/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
70
CRYSTALLIZATION NOTE
reductases are not necessarily related to one another in sequence or evolution. So far, a few studies have been reported about the reaction mechanism of the major flavin reductase of V. fischeri. Tu et al. partially purified the enzyme from V. fischeri which had no flavin and analyzed the reaction mechanism by steady-state kinetics (Tu et al., 1979). The mechanism of the enzyme catalysis was the ping-pong bisubstrate-biproduct mechanism. The enzyme was inactivated by N-ethylmaleimide in the presence of NADH, but was not inactivated alone or in the presence of FMN. Tu et al. concluded that the flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis. We have cloned the gene encoding the major NAD(P)H-flavin oxidoreductase from V. fischeri ATCC 7744 and expressed in Escherichia coli (Zenno et al., 1994). The enzyme expressed in E. coli binds FMN and is colored yellow (Inouye, 1994). Biochemical studies suggest that only bound FMN is responsible for oxidoreduction (Koike et al., unpublished data). The detailed catalytic mechanism has not been revealed. In order to gain an insight into the details of the catalytic mechanism and the substrate specificity of the enzyme, a high-resolution three-dimensional structure has to be elucidated for the enzyme. Here we report crystallization and preliminary X-ray crystallographic analysis of the major flavin reductase of V. fischeri ATCC 7744. E. coli D1210 cells (de Boer et al., 1983), carrying an FRase I expression plasmid pFR7 (Zenno et al., 1994), were cultured in LB media containing 200 µg/ml ampicillin at 37°C for 7 hr. Then cells were harvested by centrifugation at 10 000g for 30 min, and FRase I was purified as described previously (Zenno et al., 1994). The protein was purified further by loading onto a phenyl-Sepharose column (Pharma-
FIG. 1.
Plate-like yellow crystals of FRase I in a sitting drop.
71
FIG. 2. Precession photographs taken with a precession angle of 20° for 24 hr. (a) hk0 plane; systematic absences on h 1 k were observed. (b) h0l plane; systematic absences along h00 line were observed and an angle between a* and c* was 80°.
cia) in 50 mM Tris-HCl (pH 7.0) containing 20% (w/v) ammonium sulfate and subsequently eluting by a linear gradient against the same buffer without ammonium sulfate. The pure FRase I fraction was dialyzed against 5 mM Tris-HCl (pH 7.0) and concentrated to approximately 50 mg/ml using a Centricon-10 (Amicon). Crystallization was accomplished at 25°C as follows. Crystals were grown using the sitting drop vapor diffusion method by mixing equal volumes of the protein solution with a reservoir solution that contained 30% (w/v) polyethylene glycol 4000, 0.1 mM FMN, 0.2 M sodium acetate in 0.1 M Tris-HCl (pH 8.5). Plate-like yellow crystals (0.2 3 0.2 3 0.1 mm3 ) appeared over 1 month (Fig. 1). Preliminary X-ray diffraction experiments were performed using a precession camera mounted on a 4057V1 sealed tube machine (Rigaku) and an Rigaku R-AXIS IIc diffractometer equipped with imaging plates coupled to an RU-200R rotating anode generator. The sealed tube machine was operated at 40 kV and 30 mA, and a 0.5-mm collimator was used. The rotating anode was operated at 50 kV and 100 mA and focused by a double focusing mirror. To collect and process the R-AXIS data, the standard Rigaku R-AXIS software was used. Precession photographs and the R-AXIS data revealed that the crystals of FRase I belonged to the monoclinic space group C2 with unit cell dimensions, a 5 101.6 Å, b 5 63.2 Å, c 5 74.4 Å, and b 5 100.0° (Fig. 2). The assumption of one subunit per asymmetric unit leads to a Vm (crystal volume per unit of protein mass) of 4.72 Å3/Da, corresponding to a solvent content of 74% (Matthews, 1968). If two subunits exist in the asymmetric unit, a Vm value is 2.35 Å3/Da (solvent content of 48%). Because the enzyme may act as a dimer (Zenno et al., unpublished data), we expect two molecules per asymmetric unit. Intensity data were collected by using a data
72
CRYSTALLIZATION NOTE
TABLE I Statistics of the Collected X-Ray Diffraction Intensity Data of V. fischeri FRase I Resolution (Å)
Completion (%)
Intensity
Sigma
Merging R-factor
–7.14 7.14–5.05 5.05–4.13 4.13–3.58 3.58–3.20 3.20–2.92 2.92–2.70 2.70–2.53 2.53–2.38 2.38–2.26 2.26–2.16
72.2 96.3 92.9 98.2 100.0 99.1 97.7 97.1 92.6 89.8 84.1
597 675 1050 979 763 516 340 255 212 180 143
126.9 136.5 193.6 154.6 99.7 73.7 51.3 37.2 33.7 31.6 29.1
0.073 0.071 0.072 0.074 0.073 0.079 0.086 0.091 0.098 0.110 0.127
collection system at the BL6A station in the Photon Factory at the National Laboratory for High Energy Physics, by combining the Weissenberg camera for macromolecular crystallography, an imaging plate, a Fuji image reader BA100, and the data reduction program WEIS with a synchrotron radiation (Sakabe, 1983, 1991; Higashi, 1989). The crystals diffracted X-rays to at least 2.2 Å resolution and are good for a high-resolution structure determination. The data statistics are shown in Table I. In order to determine the three-dimensional structure of FRase I, the preparation of the crystals of heavyatom derivatives of the enzyme is in progress. We thank Professor N. Sakabe (Tsukuba University) for his encouragement and interest during the experiment. We also thank Dr. M. E. P. Murphy for reading the manuscript and for advice. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan. REFERENCES de Boer, H. A., Comstock, L. J., and Vasser, M. (1983) The tac promoter: A functional hybrid derived from the trp and lac promoter, Proc. Natl. Acad. Sci. USA 80, 21–25. Duane, W., and Hastings, J. W. (1975) Flavin mononucleotide reductase of luminous bacteria, Mol. Cell. Biochem. 6, 53–64. Fontecave, M., Eliasson, R., and Reichard, P. (1987) NAD(P)H: flavin oxidoreductase of Escherichia coli: A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase, J. Biol. Chem. 262, 12325–12331. Gerlo, E., and Charlier, J. (1975) Identification of NADH-specific and NADPH-specific FMN reductases in Benechea harveyi, Eur. J. Biochem. 57, 461–467. Gunsalus-Miguel, A., Meighen, E. A., Nicoli, M. Z., Nealson, K. H., and Hasting, J. W. (1972) Purification and properties of bacterial luciferase, J. Biol. Chem. 247, 398–404. Hasan, N., and Nester, E. W. (1978) Purification and characterization of NADPH-dependent flavin reductase: an enzyme required for the activation of chorismate synthase in Bacillus subtilis, J. Biol. Chem. 253, 4987–4992. Hastings, J. W., Potrikus, C. J., Gupta, S. C., Kurfurst, M., and Makemson, J. C. (1985) Biochemistry and physiology of bioluminescent bacteria, Adv. Microb. Physiol. 26, 235–291.
Hastings, J. W., Riley, W. H., and Massa, J. (1965) The purification, properties, and chemiluminescent quantum yield of bacterial luciferase, J. Biol. Chem. 240, 1473–1481. Higashi, T. (1989) The processing of diffraction data taken on a screenless Weissenberg camera for macromolecular crystallography, J. Appl. Crystallogr. 22, 9–18. Inouye, S. (1994) NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein, FEBS Lett. 347, 163–168. Jablonski, E., and DeLuca, M. (1977) Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckae harveyi, Biochemistry 16, 2932–2936. Lavi, J. T., Raunio, R. P., and Stahlberg, T. H. (1990) Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications, J. Biolumin. Chemilumin. 5, 187–192. Lo, H.-S., and Reeves, R. E. (1980) Purification and properties of NADPH:flavin oxidoreductase from Entamoeba histolytica, Mol. Biochem. Parasitol. 2, 23–30. Matthews, B. W. (1968) Solvent content of protein crystals, J. Mol. Biol. 33, 491–497. Meighen, E. A. (1991) Molecular biology of bacterial bioluminescence, Microbiol. Rev. 55, 123–142. Michaliszyn, G. A., Wing, S. S., and Meighen, E. A. (1977) Purification and properties of NAD(P)H:flavin oxidoreductase from the luminescence bacterium, Beneckea harveyi, J. Biol. Chem. 252, 7495–7499. Nefsky, B., and DeLuca, M. (1982) Studies on the NADH and NADPH:riboflavin 58-phosphate (FMN) oxidoreductases from Beneckea harveyi: Characterization of the FMN binding sites, Arch. Biochem. Biophys. 216, 10–16. Puget, K., and Michelson, A. M. (1972) Studies in bioluminescence. VII. Bacterial NADH:flavin mononucleotide oxidoreductase, Biochimie 54, 1197–1204. Sakabe, N. (1983) A focusing Weissenberg camera with multilayerline screens for macromolecular crystallography, J. Appl. Crystallogr. 16, 542–547. Sakabe, N. (1991) X-ray diffraction data collection system for modern protein crystallography with a Weissenberg camera and an imaging plate using synchrotron radiation, Nucl. Instrum. Methods A303, 448–463. Schmidt, T. M., Kopecky, K., and Nealson, K. H. (1989) Bioluminescence of insect pathogen Xenorhabdus luminescence, Appl. Envirn. Microbiol. 55, 2607–2612. Tu, S.-C., Becvar, J. E., and Hastings, J. W. (1979) Kinetic study on the mechanism of bacterial NAD(P)H:flavin oxidoreductase, Arch. Biochem. Biophys. 193, 110–116. Watanabe, H., and Hastings, J. W. (1982) Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase, Mol. Cell. Biochem. 44, 181–187. Yubisui, T., Matsuki, T., Tanishima, K., Takeshita, M., and Yoneyama, Y. (1977) NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme, Biochem. Biophys. Res. Commun. 76, 174–182. Zenno, S., and Saigo, K. (1994) Identification of the gene encoding NAD(P)H-Flavin oxidoreductase that are similar in sequence to Eschericia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harbeyi, and Vibrio orientalis, J. Bacteriol. 176, 3544–3551. Zenno, S., Saigo, K., Kanoh, H., and Inouye, S. (1994) Identification of the gene encoding the major NAD(P)H-Flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744, J. Bacteriol. 176, 3536–3543.