2-Oxo-1,2-dihydroquinoline 8-Monooxygenase, a Two-component Enzyme System from Pseudomonas putida 86

Abstract

2-Oxo-1,2-dihydroquinoline 8-monooxygenase, which catalyzes the NADH-dependent oxygenation of 2-oxo-1,2-dihydroquinoline to 8-hydroxy-2-oxo-1,2-dihydroquinoline, is the second enzyme in the quinoline degradation pathway of Pseudomonas putida 86. This enzyme system consists of two inducible protein components, which were purified, characterized, and identified as reductase and oxygenase. The yellow reductase is a monomeric iron-sulfur flavoprotein (Mr, 38,000), containing flavin adenine dinucleotide and plant-type ferredoxin [2Fe-2S]. It transferred electrons from NADH to the oxygenase or to some artificial electron acceptors. The red-brown oxygenase (Mr, 330,000) consists of six identical subunits (Mr, 55,000) and was identified as an iron-sulfur protein, possessing about six Rieske-type [2Fe-2S] clusters and additional iron. It was reduced by NADH plus catalytic amounts of reductase. For monooxygenase activity, reductase, oxygenase, NADH, molecular oxygen, and substrate were required. The activity was considerably enhanced by the addition of polyethylene glycol and Fe2+. 2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other tested compounds was converted. Based on its physical, chemical, and catalytic properties, we presume 2-oxo-1,2-dihydroquinoline 8-monooxygenase to belong to the class IB multicomponent non-heme iron oxygenases. 2-Oxo-1,2-dihydroquinoline 8-monooxygenase, which catalyzes the NADH-dependent oxygenation of 2-oxo-1,2-dihydroquinoline to 8-hydroxy-2-oxo-1,2-dihydroquinoline, is the second enzyme in the quinoline degradation pathway of Pseudomonas putida 86. This enzyme system consists of two inducible protein components, which were purified, characterized, and identified as reductase and oxygenase. The yellow reductase is a monomeric iron-sulfur flavoprotein (Mr, 38,000), containing flavin adenine dinucleotide and plant-type ferredoxin [2Fe-2S]. It transferred electrons from NADH to the oxygenase or to some artificial electron acceptors. The red-brown oxygenase (Mr, 330,000) consists of six identical subunits (Mr, 55,000) and was identified as an iron-sulfur protein, possessing about six Rieske-type [2Fe-2S] clusters and additional iron. It was reduced by NADH plus catalytic amounts of reductase. For monooxygenase activity, reductase, oxygenase, NADH, molecular oxygen, and substrate were required. The activity was considerably enhanced by the addition of polyethylene glycol and Fe2+. 2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other tested compounds was converted. Based on its physical, chemical, and catalytic properties, we presume 2-oxo-1,2-dihydroquinoline 8-monooxygenase to belong to the class IB multicomponent non-heme iron oxygenases. The soil bacterium Pseudomonas putida 86 utilizes quinoline as sole source of carbon, nitrogen, and energy. The occurrence of the metabolites 2-oxo-1,2-dihydroquinoline, 8-hydroxy-2-oxo-1,2-dihydroquinoline, 8-hydroxycoumarin, and 2,3-dihydroxyphenylpropionic acid revealed that the degradation of quinoline proceeds in this organism via the so called “coumarin pathway”(1Schwarz G. Bauder R. Speer M. Rommel T.O. Lingens F. Biol. Chem. Hoppe-Seyler. 1989; 370: 1183-1189Google Scholar, 2Shukla O.P. Appl. Environ. Microbiol. 1986; 51: 1332-1342Google Scholar). Quinoline 2-oxidoreductase from this organism, catalyzing the initial nucleophilic attack of quinoline at C-2, yielding 2-oxo-1,2-dihydroquinoline, is investigated thoroughly(3Bauder R. Tshisuaka B. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 1137-1144Google Scholar, 4Tshisuaka B. Kappl R. Httermann J. Lingens F. Biochemistry. 1993; 32: 12928-12934Google Scholar). However, no enzymatic study is available concerning the further conversions of 2-oxo-1,2-dihydroquinoline in the “coumarin pathway.” In this paper, we present the purification and characterization of 2-oxo-1,2-dihydroquinoline 8-monooxygenase. This novel two-component enzyme system is compared with other multicomponent enzymes belonging to the family of non-heme iron oxygenases. DEAE-cellulose DE-52 was obtained from Whatman (Maidstone, Great Britain); phenyl-Sepharose CL-4B, Sephacryl S-300, Superose 12 HR 10/30, and Superdex 200 prep grade HiLoad 16/60 were from Pharmacia Biotech (Freiburg, Germany); DEAE-Fractogel EMD was obtained from Merck (Darmstadt, Germany); HPLC columns were from Bischoff (Leonberg, Germany); YM-10 and YM-30 ultrafiltration membranes were purchased from Amicon (Witten, Germany); and PEG1 1The abbreviations used are: PEGpolyethylene glycolDCPIP2,6-dichlorophenol indophenolgavaverage value of the g-tensor gav = 1/3 (g + g + g)HPLChigh pressure liquid chromatographyINT2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride. 6000 and carrier ampholytes (Servalyt 3-6) were obtained from Serva (Heidelberg, Germany). Quinoline and 2-oxo-1,2-dihydroquinoline were a gift from Ruetgerswerke AG (Castrop Rauxel, Germany). 8-Hydroxy-2-oxo-1,2-dihydroquinoline was obtained by biotransformation.2 2B. Tshisuaka, unpublished results. All other chemicals and biochemicals were of the highest purity commercially available. polyethylene glycol 2,6-dichlorophenol indophenol average value of the g-tensor gav = 1/3 (g + g + g) high pressure liquid chromatography 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride. P. putida 86 was described by Schwarz et al.(5Schwarz G. Senghas E. Erben A. Schfer B. Lingens F. Hke H. Syst. Appl. Microbiol. 1988; 10: 185-190Google Scholar). The strain was grown aerobically at 30°C on mineral salts medium containing (g/liter) 4.3 Na2HPO4× 2 H2O, 1.15 KH2PO4, 1.0 NaCl, 0.4 MgSO4× 7 H2O, 0.05 FeSO4× 7 H2O, and 0.01 Na2MoO4× 2 H2O. After sterilization, 4 mM quinoline was added. For studies of enzyme induction, the medium contained (instead of quinoline) 2.8 mM 2-oxo-1,2-dihydroquinoline or glucose (2 g/liter) plus (NH4)2SO4 (1 g/liter). After 15 h of growth, cells were washed twice with 50 mM Tris-HCl buffer (pH 7.5) and suspended (adjusting A578 to 2.2) in fresh mineral salts medium containing (i) 2.8 mM 2-oxo-1,2-dihydroquinoline or (ii) 2.8 mM 2-oxo-1,2-dihydroquinoline plus 50 mg/liter chloramphenicol. Degradation of 2-oxo-1,2-dihydroquinoline was monitored spectrophotometrically at 323 nm. Large-scale growth of bacteria was carried out in a 100-liter fermenter with quinoline as substrate. Bacterial growth and concentrations of quinoline and 2-oxo-1,2-dihydroquinoline were measured spectrophotometrically according to Bauder et al.(3Bauder R. Tshisuaka B. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 1137-1144Google Scholar). After 9, 23, 32, and 48 h of fermentation, bacteria were fed with 4 mM quinoline. After total consumption of quinoline and 2-oxo-1,2-dihydroquinoline, cells were harvested by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 7.5), and stored at −20°C. All purification steps were carried out at 4°C, except for fast protein liquid chromatography (DEAE-Fractogel EMD and Superose 12), which was performed at room temperature. All pH values given refer to room temperature. In Step 1, frozen cells (40 g, wet weight) were thawed in 40 ml of standard buffer (50 mM Tris-HCl, pH 7.3) and disrupted 5 min in time intervals of 0.5 s in a sonifier (model 450, Branson Inc., Danbury, CT) with maximal power. The suspension was centrifuged at 48,000 × g for 40 min. In Step 2, the supernatant was diluted with the same volume of standard buffer and loaded for anion-exchange chromatography onto a DEAE-cellulose DE-52 column (2.5 × 39 cm) equilibrated in standard buffer. The column was washed with the same buffer, and bound proteins were desorbed with a linear gradient (800 ml) of KCl (0-0.6 M) in standard buffer at a flow rate of 0.6 ml/min. This purification step largely separated the reductase from the oxygenase. In Step 3, fractions containing the reductase were combined and adjusted to 0.8 M (NH4)2SO4. For hydrophobic interaction chromatography, the solution was applied to a column of phenyl-Sepharose (2.5 × 5 cm) equilibrated in 50 mM Tris-HCl buffer, pH 7.5, containing 0.8 M (NH4)2SO4 and 1 mM dithiothreitol. The column was rinsed with 200 ml of the same buffer, and proteins were eluted with a linear gradient (300 ml) of 0.8-0 M (NH4)2SO4, 50-10 mM Tris-HCl (pH 7.5), 0-10% (v/v) ethanol, containing 1 mM dithiothreitol. The flow rate was 3 ml/min. The combined fractions with reductase activity were rinsed with 50 mM Tris-HCl buffer, pH 7.8, containing 1 mM dithiothreitol, by ultrafiltration with a YM-10 membrane. In Step 4, for a second anion-exchange chromatography, the concentrated protein solution was applied to a DEAE-Fractogel EMD column (1 × 14 cm) equilibrated in 50 mM Tris-HCl buffer, pH 7.8, containing 1 mM dithiothreitol. The column was rinsed with 3 ml of the same buffer, and a linear gradient (35 ml) of 0-0.5 M (NH4)2SO4 was applied at a flow rate of 1 ml/min. Active fractions were pooled and concentrated to 0.5 ml by ultrafiltration with a YM-10 membrane. In Step 5, for gel filtration, the protein solution was loaded onto Superose 12 (1.5 × 28 cm), which was equilibrated in 100 mM Tris-HCl buffer, pH 7.8, containing 1 mM dithiothreitol. Proteins were separated at a flow rate of 0.5 ml/min. Fractions containing the reductase were pooled and stored at −80°C. Preparation of crude extract (Step 1′) and chromatography on DEAE-cellulose DE-52 (Step 2′) were performed as described above for the reductase, except that a crude extract from 20 g (wet weight) of cells was used. In Step 3′, fractions from DEAE-cellulose DE-52 containing the oxygenase were pooled and adjusted to 0.6 M (NH4)2SO4 and 3% (w/v) glycerol. The solution was loaded on a column of phenyl-Sepharose (2.5 × 5 cm), equilibrated in 50 mM Tris-HCl buffer, pH 7.5, containing 0.6 M (NH4)2SO4, 3% (w/v) glycerol, and 0.1 mM 2-oxo-1,2-dihydroquinoline. The column was washed with the same buffer, and the oxygenase was eluted by omitting (NH4)2SO4 from the buffer. To avoid severe losses of oxygenase activity, fast performance of all steps demanding high (NH4)2SO4 concentrations was substantial (flow rate, 10 ml/min). In Step 4′, after concentrating the eluate by ultrafiltration (YM-30 membrane), it was passed through a Sephacryl S-300 gel filtration column (2.5 × 92 cm; flow rate, 0.5 ml/min) using standard buffer containing 50 mM KCl and 0.1 mM 2-oxo-1,2-dihydroquinoline. In Step 5′, for adsorption chromatography on hydroxyapatite, which was prepared by the method of Atkinson et al.(6Atkinson A. Bradford P.A. Selmes I.P. J. Appl. Chem. Biotechnol. 1973; 23: 517-529Google Scholar), fractions with oxygenase activity were combined and adjusted to pH 7.2 with 1 M acetate buffer, pH 5.0. Glycerol was included to a final concentration of 3% (w/v), and 1 M potassium phosphate buffer, pH 7.2, was added to a final concentration of 10 mM. The solution was applied to a column of hydroxyapatite (2.5 × 5 cm), equilibrated in 10 mM potassium phosphate buffer, pH 7.2, containing 3% (w/v) glycerol and 0.1 mM 2-oxo-1,2-dihydroquinoline. The column was washed with the same buffer. Proteins were eluted with a linear gradient (300 ml) of 10-300 mM potassium phosphate buffer, pH 7.2 (3% (w/v) glycerol and 0.1 mM 2-oxo-1,2-dihydroquinoline), at a flow rate of 0.8 ml/min. For reproducible results, hydroxyapatite was used only once. Fractions containing the oxygenase were pooled, concentrated by ultrafiltration with a YM-30 membrane, and stored at −80°C. Since the oxygenase lost 50% specific activity when stored in standard buffer for 3 days at 4°C, different stabilizers were tested: 10% (w/v) glycerol, 10% (v/v) ethanol, 0.2% Triton X-100, 0.1 mM EDTA, 1-10 mM mercaptoethanol, 1 mM glutathione, 0.1-1 mM dithiothreitol, 0.1-1 mM (NH4)2Fe(SO4)2, and 0.1 mM 2-oxo-1,2-dihydroquinoline. Further attempts to preserve enzyme activity were made by using other buffers (pH 6-8.5) or purification procedures such as precipitation with (NH4)2SO4, PEG, or ethanol, chromatography on Superdex 200, chelating Sepharose, calcium tartrate or Mono-Q (anion exchange), and preparative native gel electrophoresis. Progress in enzyme purification was monitored by SDS-polyacrylamide gel electrophoresis with 10% separating and 4% stacking gels(7Schgger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Google Scholar). The 1-mm slab gels were stained in 0.2% (w/v) Coomassie Blue R-250 in water/methanol/acetic acid (40:50:10). Analytical isoelectric focusing was performed in rehydrated gels as recommended by Pharmacia(8Schickle H. Westermeier R. Pharmacia Bulletin. Freiburg, Germany1987Google Scholar). The native molecular weight of the reductase was determined by gel filtration through Superose 12 in 50 mM Tris-HCl buffer, pH 7.8, containing 150 mM NaCl. The native molecular weight of the oxygenase was estimated by gel filtration on Superdex 200 in 100 mM Tris-HCl buffer, pH 7.5. The molecular weights of peptides under denaturing conditions were determined using SDS-polyacrylamide gel electrophoresis(7Schgger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Google Scholar). Absorption spectra were measured in cells of 1-cm path length at 25°C with a Uvicon 930 spectrometer (Kontron Instruments, Neufahrn, Germany). The flavin cofactor was extracted from the reductase by two methods. The component in 10 mM potassium phosphate buffer (pH 7.0) was boiled for 4 min or treated with trichloroacetic acid as described by Siegel(9Siegel L.M. Methods Enzymol. 1978; 53: 419-429Google Scholar). After removal of protein by centrifugation and ultrafiltration, the yellow filtrate was examined by UV-visible spectroscopy (ϵFAD, 450 nm, 11,300 M-1 cm-1(10Whitby L.G. Biochem. J. 1953; 54: 437-442Google Scholar)) and by reversed phase HPLC on a Lichrospher RP18 column according to Nielsen et al.(11Nielsen P. Rauschenbach P. Bacher A. Methods Enzymol. 1986; 122: 209-220Google Scholar). 0.2 M formic acid, 0.1 M ammonia/methanol (3:1) was used as eluent. Acid-labile sulfur was determined by the formation of methylene blue as described by Beinert(12Beinert H. Anal. Biochem. 1983; Scholar). was used as a of was with an spectrometer Inc., concentrations were determined according to Bradford Anal. Biochem. with as The activity of 2-oxo-1,2-dihydroquinoline 8-monooxygenase was measured at 25°C spectrophotometrically at as NADH consumption M-1 Chem. Biochem. Scholar)) or as with a The was for and buffer as as for the concentrations of buffer, PEG, NADH, and 2-oxo-1,2-dihydroquinoline. The mM Tris-HCl buffer (pH 7.5) contained PEG, 0.05 mM (NH4)2Fe(SO4)2, mM NADH, and amounts of reductase and oxygenase The was by the addition of 0.2 mM 2-oxo-1,2-dihydroquinoline. All were for NADH or consumption in the of substrate. 1 of 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity was as the of enzyme that 1 of NADH or 1 of min. The enzyme activity was to the crude extract concentration in the To a the activity of the reductase was measured in the of an of the oxygenase component and to added by the purification steps 1 and were stored at consumption and formation were monitored by HPLC RP18 (v/v) with UV-visible at nm. was further by chromatography of acetate of the gel acid G. Bauder R. Speer M. Rommel T.O. Lingens F. Biol. Chem. Hoppe-Seyler. 1989; 370: 1183-1189Google as in The contained of reductase, of oxygenase, 0.05 mM (NH4)2Fe(SO4)2, 2 mM NADH, and 2 mM 2-oxo-1,2-dihydroquinoline in 50 mM Tris-HCl buffer (pH of other was monitored activity under conditions by and with was examined by chromatography as described reductase activity was at 25°C spectrophotometrically as of the artificial electron M-1 Chem. 1973; M-1 M-1 and potassium M-1 J. Biol. Chem. The contained mM Tris-HCl buffer (pH 7.5), 0.05 mM electron and mM The was by the addition of of protein 1 of reductase activity was as the of enzyme that reduced 1 of electron min. The of on reductase activity and on 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity were examined in the of Fe2+. were 2 min with The protein concentrations were 14 in the reductase and in the standard of P. putida grown on glucose as sole source of and 2-oxo-1,2-dihydroquinoline in the of an of protein cells 2-oxo-1,2-dihydroquinoline after a phase of 2 grown on 2-oxo-1,2-dihydroquinoline, this substrate when added by the addition of chloramphenicol. the degradation of 2-oxo-1,2-dihydroquinoline was on protein by 2-oxo-1,2-dihydroquinoline. of grown on glucose or reductase oxygenase activity of 2-oxo-1,2-dihydroquinoline 8-monooxygenase. cells were grown on 2-oxo-1,2-dihydroquinoline, a specific reductase activity of protein and a specific oxygenase activity of protein was measured in the we that of 2-oxo-1,2-dihydroquinoline 8-monooxygenase in P. putida 86 inducible by the substrate 2-oxo-1,2-dihydroquinoline. The of a purification in The reductase was in a of The oxygenase activity was only However, as by SDS-polyacrylamide gel a high of the oxygenase protein in to other proteins was by the gels of the oxygenase and reductase that the in specific activity that the oxygenase lost activity the purification it with glycerol and substrate. tested stabilizers or purification procedures were in enzyme in a The of reductase and oxygenase were as by SDS-polyacrylamide gel electrophoresis The native molecular weight of reductase was determined to by gel molecular weight determined under denaturing and conditions by SDS-polyacrylamide gel electrophoresis was we the reductase to a filtration of the oxygenase in a native molecular weight of Since SDS-polyacrylamide gel electrophoresis of the oxygenase revealed only protein to a molecular weight of we that the oxygenase is of six identical The isoelectric of the reductase was The oxygenase, which was as by SDS-polyacrylamide gel separated in isoelectric focusing as a of the of pH by other R. J. Scholar, Methods Enzymol. Scholar), the formation of an to formation of proteins with carrier In the the reductase was yellow in The UV-visible at and nm, a at and nm, and at about and The reductase was reduced and by the addition of After and a reductase the supernatant an 2, for a of oxygenase, as in the a red-brown The UV-visible at and and a at of the oxygenase in the of The oxygenase as was by with or by with NADH and catalytic amounts of reductase. The addition of NADH the oxygenase. This that the reductase, which was reduced by NADH, the electron from NADH to the oxygenase by HPLC revealed an of of of reductase. of the in a of of of reductase. of g iron and g sulfur were determined of reductase. 1 of oxygenase contained g iron and g The spectra that the iron is bound to a is of iron-sulfur studies revealed that a plant-type ferredoxin [2Fe-2S] with gav = is present in the reductase the oxygenase component Rieske-type [2Fe-2S] clusters = F. and A. for that the reductase and plant-type ferredoxin [2Fe-2S] and that the oxygenase six Rieske-type [2Fe-2S] clusters [2Fe-2S] and additional iron. oxygenase, NADH, molecular oxygen, and substrate were for 2-oxo-1,2-dihydroquinoline 8-monooxygenase of with concentrations of 2-oxo-1,2-dihydroquinoline or NADH revealed a for the highest specific under conditions of the standard of and were catalytic activity was under The pH of the enzyme activity was pH 7.5. The was in the of The reductase component of 2-oxo-1,2-dihydroquinoline 8-monooxygenase reductase activity only toward the oxygenase component of this enzyme system toward some artificial electron acceptors. with a specific activity of 40 measured in the of the oxygenase component specific of and with and The oxygenase no activity with acceptors. In the reductase the reductase only activity NADH was by The addition of and to an in 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity that lost and was as electron NADH of the enzyme with and further in a of the and (50 in its However, the addition of 50 or in or of activity, compared to the activity In to the 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity, the reductase activity was by the addition of or The reductase was by and and it was by 2-Oxo-1,2-dihydroquinoline the reductase on 2-oxo-1,2-dihydroquinoline 8-monooxygenase was by the addition of to the enzyme In the of PEG, the was only of activity, which to 12 min as determined in the of However, reductase activity was in the of that and 0.5 mM reductase activity and and the activity of the monooxygenase system and chelating acid, and 0.5 mM the reductase activity, the enzyme system was and 8-hydroxy-2-oxo-1,2-dihydroquinoline, and mM consumption converted. consumption was with quinoline acid, and mM inducible two-component enzyme which catalyzes the NADH-dependent of 2-oxo-1,2-dihydroquinoline to 8-hydroxy-2-oxo-1,2-dihydroquinoline, was from P. putida 86. The enzyme system was 2-oxo-1,2-dihydroquinoline 8-monooxygenase with the 2-oxo-1,2-dihydroquinoline, 2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other compounds tested was converted. However, 8-hydroxy-2-oxo-1,2-dihydroquinoline, and consumption that compounds as electron from substrate with of This for some other Scholar), H. J. Biol. Chem. Scholar), or H. J. Biochem. 1973; Scholar). Since added under in of protein, was in the enzyme that 2-oxo-1,2-dihydroquinoline 8-monooxygenase is a multicomponent enzyme system was the enzyme activity and protein The specific enzyme activity with protein of Since of the two enzyme is a for monooxygenase activity, a the time of activity in the of The of enzyme by on the component to its To from this other multicomponent enzyme tested for the of 4 a for the electron from NADH to the substrate oxygenase. NADH two electrons as iron-sulfur to The of the reductase is to this by its flavin The oxygenase its Rieske-type iron-sulfur clusters additional iron. The of iron for the catalytic of the oxygenase component is by the that chelating 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity, reductase activity was This with the that added iron reductase activity, the enzyme was we that iron is a cofactor of the oxygenase which is by chelating or enzyme purification and which by added iron. to 2-oxo-1,2-dihydroquinoline 8-monooxygenase to the of non-heme iron multicomponent which iron-sulfur clusters and additional iron in oxygenase In to component or multicomponent which by or Biotechnol. 1989; Scholar), non-heme iron to by R. Microbiol. Scholar). In the of the oxygenase component of the monooxygenase non-heme iron was to the electron from a Rieske-type [2Fe-2S] to molecular oxygen, the as for the attack of the E. E. A. H. Chem. Scholar, E. J. Biochem. Scholar). This for all multicomponent non-heme iron which Rieske-type iron-sulfur clusters and iron in oxygenase In Biochem. J. Scholar, J. Scholar, H. H. J. Biochem. Scholar, E. J. Biochem. 1978; Scholar, A. Lingens F. J. Biol. Chem. 1986; Scholar, A. M. Lingens F. J. Biol. Chem. 1987; Scholar, E. J. Biol. Chem. 1987; Scholar, F. J. J. 1988; Scholar, R. Lingens F. J. Scholar, B. Lingens F. J. Scholar, M. H. J. Biol. Chem. 1978; Scholar, M. H. J. Biol. Chem. Scholar, M. H. J. Biol. Chem. Scholar, J. 1993; Scholar, G. J. Lingens F. J. Biochem. Scholar, F. R. Biochem. J. Scholar, Biochem. J. Scholar, Biochem. Scholar, M. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J. Scholar, J. 1983; Scholar, J. 1990; Scholar, J. 1990; Scholar, H. J. Scholar), 2-oxo-1,2-dihydroquinoline 8-monooxygenase is compared with some of multicomponent non-heme iron oxygenases. et F. and of the and on the of protein and on the in electron from to the oxygenase two a flavin and a [2Fe-2S] in this electron second [2Fe-2S] is The on or two protein components, or enzyme enzymes two-component enzyme with a flavin in class in class and a plant-type ferredoxin [2Fe-2S] in the reductase. In class the flavin and the [2Fe-2S] on The [2Fe-2S] is a plant-type ferredoxin or a Rieske-type enzymes and plant-type ferredoxin in the reductase as as a Rieske-type in a second to this 2-oxo-1,2-dihydroquinoline 8-monooxygenase to class IB enzyme as R. Lingens F. J. Scholar, B. Lingens F. J. Scholar), M. H. J. Biol. Chem. 1978; Scholar, M. H. J. Biol. Chem. Scholar, M. H. J. Biol. Chem. Scholar), and M. 1986; Scholar). However, the oxygenase of class IB enzymes 2-oxo-1,2-dihydroquinoline 8-monooxygenase is a the class enzymes in in a 2-Oxo-1,2-dihydroquinoline 8-monooxygenase to other multicomponent non-heme iron J. Scholar), J. J. 1990; Scholar), or M. M. J. Scholar, J. Biochem. Scholar), the oxygenase of enzyme iron-sulfur 2-oxo-1,2-dihydroquinoline 8-monooxygenase is a monooxygenase or the is to of an or of 2-oxo-1,2-dihydroquinoline, a is it to determined is an in in the “coumarin of quinoline degradation by P. putida 86. However, no was the enzyme in under the of a was from the substrate by cells of P. putida B. Lingens F. Microbiol. 1993; Scholar). This activity is to from 2-oxo-1,2-dihydroquinoline 8-monooxygenase the enzyme system This the of enzymes in Schwarz et G. Bauder R. Speer M. Rommel T.O. Lingens F. Biol. Chem. Hoppe-Seyler. 1989; 370: 1183-1189Google described two of quinoline In the step of quinoline is to 2-oxo-1,2-dihydroquinoline, by quinoline which a from R. Tshisuaka B. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 1137-1144Google Scholar, B. Lingens F. Biol. Chem. Hoppe-Seyler. Scholar). the quinoline from bacteria R. Tshisuaka B. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 1137-1144Google Scholar, B. Lingens F. Biol. Chem. Hoppe-Seyler. Scholar, B. Tshisuaka B. Lingens F. Biol. Chem. Hoppe-Seyler. Scholar), 4 B. Tshisuaka, and F. for the catalyzing the degradation step attack the same substrate at different The enzyme of from the enzyme system of P. putida at The of nucleophilic as as a to for with Inc., Scholar). to A. for electron for and spectra and for