Oxygenases without requirement for cofactors or metal ions.

Mono- and dioxygenases usually depend on a transition metal or an organic cofactor to activate dioxygen, or their organic substrate, or both. This review points out that there are at least two separate families of oxygenases without any apparent requirement for cofactors or metal ions: the quinone-forming monooxygenases which are important 'tailoring enzymes' in the biosynthesis of several types of aromatic polyketide antibiotics, and the bacterial dioxygenases involved in the degradation of distinct quinoline derivatives, catalyzing the 2,4-dioxygenolytic cleavage of 3-hydroxy-4-quinolones with concomitant release of carbon monoxide. The quinone-forming monooxygenases might be useful for the modification of polyketide structures, either by using them as biocatalysts, or by employing combinatorial biosynthesis approaches. Cofactor-less oxygenases present the mechanistically intriguing problem of how dioxygen is activated for catalysis. However, the reactions catalyzed by these enzymes are poorly understood in mechanistic terms. Formation of a protein radical and a substrate-derived radical, or direct electron transfer from a deprotonated substrate to molecular oxygen to form a caged radical pair may be discussed as hypothetical mechanisms. The latter reaction route is expected for substrates that can easily donate an electron to dioxygen, and requires the ability of the enzyme to stabilize anionic intermediates. Histidine residues found to be catalytically relevant in both types of cofactor-less oxygenases might be involved in substrate deprotonation and/or electrostatic stabilization.

Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization.

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.

Site-directed mutagenesis of potential catalytic residues in 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase, and hypothesis on the catalytic mechanism of 2,4-dioxygenolytic ring cleavage.

1H-3-Hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) is a cofactor-free dioxygenase proposed to belong to the alpha/beta hydrolase fold superfamily of enzymes. Alpha/beta Hydrolases contain a highly conserved catalytic triad (nucleophile-acidic residue-histidine). We previously identified a corresponding catalytically essential histidine residue in Qdo. However, as shown by amino acid replacements through site-directed mutagenesis, nucleophilic and acidic residues of Qdo considered as possible triad residues were not absolutely required for activity. This suggests that Qdo does not contain the canonical catalytic triad of the alpha/beta hydrolase fold enzymes. Some radical trapping agents affected the Qdo-catalyzed reaction. A hypothetical mechanism of Qdo-catalyzed dioxygenation of 1H-3-hydroxy-4-oxoquinoline is compared with the dioxygenation of FMNH2 catalyzed by bacterial luciferase, which also uses a histidine residue as catalytic base.

Enzymes involved in the aerobic bacterial degradation of N-heteroaromatic compounds: molybdenum hydroxylases and ring-opening 2,4-dioxygenases.

Many N-heteroaromatic compounds are utilized by micro-organisms as a source of carbon (and nitrogen) and energy. The aerobic bacterial degradation of these growth substrates frequently involves several hydroxylation steps and subsequent dioxygenolytic cleavage of (di)hydroxy-substituted heteroaromatic intermediates to aliphatic metabolites which finally are channeled into central metabolic pathways. As a rule, the initial bacterial hydroxylation of a N-heteroaromatic compound is catalyzed by a molybdenum hydroxylase, which uses a water molecule as source of the incorporated oxygen. The enzyme's redox-active centers - the active site molybdenum ion coordinated to a distinct pyranopterin cofactor, two different [2Fe2S] centers, and in most cases, flavin adenine dinucleotide - transfer electrons from the N-heterocyclic substrate to an electron acceptor, which for many molybdenum hydroxylases is still unknown. Ring-opening 2,4-dioxygenases involved in the bacterial degradation of quinaldine and 1H-4-oxoquinoline catalyze the cleavage of two carbon-carbon bonds with concomitant formation of carbon monoxide. Since they contain neither a metal center nor an organic cofactor, and since they do not show any sequence similarity to known oxygenases, these unique dioxygenases form a separate enzyme family. Quite surprisingly, however, they appear to be structurally and mechanistically related to enzymes of the alpha/beta hydrolase fold superfamily. Microbial enzymes are a great resource for biotechnological applications. Microbial strains or their enzymes may be used for degradative (bioremediation) or synthetic (biotransformation) purposes. Modern bioremediation or biotransformation strategies may even involve microbial catalysts or strains designed by protein engineering or pathway engineering. Prerequisite for developing such modern tools of biotechnology is a comprehensive understanding of microbial metabolic pathways, of the structure and function of enzymes, and of the molecular mechanisms of biocatalysis.

Kinetics and interactions of molybdenum and iron-sulfur centers in bacterial enzymes of the xanthine oxidase family: mechanistic implications.

For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.

Bacterial 2,4-dioxygenases: new members of the alpha/beta hydrolase-fold superfamily of enzymes functionally related to serine hydrolases.

1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from Pseudomonas putida 33/1 and 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from Arthrobacter ilicis Rü61a catalyze an N-heterocyclic-ring cleavage reaction, generating N-formylanthranilate and N-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the alpha/beta hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 strongly suggested that Qdo and Hod are structurally related to the alpha/beta hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the alpha/beta hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed.

Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein.

Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carbon monoxide and 2-protocatechuoyl-phloroglucinol carboxylic acid, was purified from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glycan chains. The enzyme was resolved in SDS polyacrylamide gels in a diffuse protein band that corresponded to a molecular mass of 130-170 kDa. When purified flavonol 2,4-dioxygenase was heated, it dissociated into three peptides with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-35 kDa (S), which occurred in a molar ratio of 1:1:1, suggesting a LMS structure. Crosslinking led to a 90-97 kDa species, concomitant with the decrease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) peptides. Analysis by matrix-assisted laser desorption/ionization-time of flight-MS showed peaks at m/z approximately 69 600, m/z approximately 51 700, and m/z approximately 26 500 which are presumed to represent the three peptides of flavonol 2,4-dioxygenase, and a broad peak at m/z approximately 96 300, which might correspond to the LS heterodimer as formed in the crosslinking reaction. Based on the estimated molecular mass of 148 kDa, 1 mol of enzyme contained 1.0-1.6 mol of copper. Ethylxanthate, which specifically reduces CuII to CuI ethylxanthate, is a potent inhibitor of flavonol 2,4-dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphenylthiocarbazone) strongly inhibited the enzymatic activity, but inactivation was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-dioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 CuII protein. The Cu2+ is assumed to interact with four nitrogen ligands, and the CuII complex has a (distorted) square planar geometry.

Bioconversion of pyrimidine by resting cells of quinoline-degrading bacteria.

Nine quinoline-degrading bacterial strains were tested for their ability to hydroxylate pyrimidine. All strains converted pyrimidine to uracil via pyrimidine-4-one in a cometabolic process. Quinoline 2-oxidoreductases (QuinORs) were the catalysts of fortuitous pyrimidine hydroxylation. Whereas in most strains the activity of the QuinOR towards pyrimidine was very low compared to its activity towards quinoline, QuinOR in crude extracts from Comamonas testosteroni 63 showed a specific activity of 64 (mU mg protein)-1 with pyrimidine as substrate, compared to a specific activity of 237 (mU mg protein)-1 towards the intrinsic substrate quinoline. Resting cells of Comamonas testosteroni 63 rapidly converted pyrimidine almost stoichiometrically to uracil, which accumulated in the cell suspension. Using an adsorbent resin, uracil was prepared from the supernatant of Comamonas testosteroni 63 resting cells with a yield of > 98%.

Cloning, sequence analysis, and expression of the Pseudomonas putida 33/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase gene, encoding a carbon monoxide forming dioxygenase.

1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from the 1H-4-oxoquinoline utilizing Pseudomonas putida strain 33/1, which catalyzes the cleavage of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilate, is devoid of any transition metal ion or other cofactor and thus represents a novel type of ring-cleavage dioxygenase. Gene qdo was cloned and sequenced. Its overexpression in Escherichia coli yielded recombinant His-tagged Qdo which was catalytically active. Qdo exhibited 36% and 16% amino acid identity to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) and atropinesterase (a serine hydrolase), respectively. Qdo as well as Hod possesses a SXSHG motif, resembling the motif GXSXG of the serine hydrolases which comprises the active-site nucleophile (X=arbitrary residue).

Bacterial dehalogenation.

Halogenated organic compounds are produced industrially in large quantities and represent an important class of environmental pollutants. However, an abundance of haloorganic compounds is also produced naturally. Bacteria have evolved several strategies for the enzyme-catalyzed dehalogenation and degradation of both haloaliphatic and haloaromatic compounds: (i) Oxidative dehalogenation is the result of mono- or dioxygenase-catalyzed, co-metabolic or metabolic reactions. (ii) In dehydrohalogenase-catalyzed dehalogenation, halide elimination leads to the formation of a double bond. (iii) Substitutive dehalogenation in most cases is a hydrolytic process, catalyzed by halidohydrolases, but there also is a "thiolytic" mechanism with glutathione as cosubstrate. Dehalogenation by halohydrin hydrogen-halide lyases is the result of an intramolecular substitution reaction. (iv) A distinct dechlorination mechanism involves methyl transfer from chloromethane onto tetrahydrofolate. (v) Reductive dehalogenations are co-metabolic processes, or they are specific reactions involved in substrate utilization (carbon metabolism), or reductive dehalogenation is coupled to energy conservation: some anaerobic bacteria use a specific haloorganic compound as electron acceptor of a respiratory process. This review discusses the mechanisms of enzyme-catalyzed dehalogenation reactions, describes some pathways of the bacterial degradation of haloorganic compounds, and indicates some trends in the biological treatment of organohalogen-polluted air, groundwater, soil, and sediments.

Comparative EPR and redox studies of three prokaryotic enzymes of the xanthine oxidase family: quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase.

For three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase, the electron transfer centers were investigated by electron paramagnetic resonance. The enzymes are containing a molybdenum-molybdopterin cytosine dinucleotide cofactor, two distinct [2Fe-2S] clusters and, apart from isoquinoline 1-oxidoreductase, a flavin adenine dinucleotide. The latter cofactor yields two different organic radical signals in quinoline 2-oxidoreductase and quinaldine 4-oxidase, typical for the neutral and anionic form, respectively. A "rapid" Mo(V) species is present in all enzymes with small differences in magnetic parameters. From spectra simulation of 95Mo-substituted quinoline 2-oxidoreductase, a deviation of 25 degrees between the maximal g and 95Mo-hyperfine tensor component was derived. The very rapid Mo(V) species was detected in small amounts upon reduction with substrates in quinoline 2-oxidoreductase and quinaldine 4-oxidase, but showed a different kinetic behavior with considerable EPR intensities in isoquinoline 1-oxidoreductase. The FeSI and FeSII centers produced different signals in all three enzymes and, in case of isoquinoline 1-oxidoreductase, revealed a dipolar interaction, from which a maximum distance of 15 A between FeSI and FeSII was estimated. The midpoint potentials of the FeS centers were surprisingly different and determined for FeSI/FeSII with -155/-195 mV in quinoline 2-oxidoreductase, -250/-70 mV in quinaldine 4-oxidase, and +65/+10 mV in isoquinoline 1-oxidoreductase. The slopes of the fitting curves for the Nernst equation are indicative for nonideal behavior. Only in quinoline 2-oxidoreductase, an averaged midpoint potential of the molybdenum redox pairs of about -390 mV could be determined. Both of the other enzymes did not produce Mo(V) signals in redox titration experiments, probably because of direct reduction of Mo(VI) to Mo(IV) in the presence of dithionite.

2-oxo-1,2-dihydroquinoline 8-monooxygenase: phylogenetic relationship to other multicomponent nonheme iron oxygenases.

2-Oxo-1,2-dihydroquinoline 8-monooxygenase, an enzyme involved in quinoline degradation by Pseudomonas putida 86, had been identified as a class IB two-component nonheme iron oxygenase based on its biochemical and biophysical properties (B. Rosche, B. Tshisuaka, S. Fetzner, and F. Lingens, J. Biol. Chem. 270:17836-17842, 1995). The genes oxoR and oxoO, encoding the reductase and the oxygenase components of the enzyme, were sequenced and analyzed. oxoR was localized approximately 15 kb downstream of oxoO. Expression of both genes was detected in a recombinant Pseudomonas strain. In the deduced amino acid sequence of the NADH:(acceptor) reductase component (OxoR, 342 amino acids), putative binding sites for a chloroplast-type [2Fe-2S] center, for flavin adenine dinucleotide, and for NAD were identified. The arrangement of these cofactor binding sites is conserved in all known class IB reductases. A dendrogram of reductases confirmed the similarity of OxoR to other class IB reductases. The oxygenase component (OxoO, 446 amino acids) harbors the conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and the mononuclear iron. In contrast to known class IB oxygenase components, which are composed of differing subunits, OxoO is a homomultimer, which is typical for class IA oxygenases. Sequence comparison of oxygenases indeed revealed that OxoO is more related to class IA than to class IB oxygenases. Thus, 2-oxo-1,2-dihydroquinoline 8-monooxygenase consists of a class IB-like reductase and a class IA-like oxygenase. These results support the hypothesis that multicomponent enzymes may be composed of modular elements having different phylogenetic origins.

2,4-dioxygenases catalyzing N-heterocyclic-ring cleavage and formation of carbon monoxide. Purification and some properties of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase from Arthrobacter sp. Rü61a and comparison with 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase from Pseudomonas putida 33/1.

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (MeQDO) was purified from quinaldine-grown Arthrobacter sp. Rü61a. It was enriched 59-fold in a yield of 22%, and its properties were compared with 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) purified from Pseudomonas putida 33/1. The enzyme-catalyzed conversions were performed in an (18O)O2/(16O)O2 atmosphere. Two oxygen atoms of either (18O)O2 or (16O)O2 were incorporated at C2 and C4 of the respective substrates, indicating that these unusual enzymes, which catalyze the cleavage of two carbon-carbon bonds concomitant with CO formation, indeed are 2,4-dioxygenases. Both enzymes are small monomeric proteins of 32 kDa (MeQDO) and 30 kDa (QDO). The apparent K(m) values of MeQDO for 1H-3-hydroxy-4-oxoquinaldine and QDO for 1H-3-hydroxy-4-oxoquinoline were 30 microM and 24 microM, respectively. In both 2,4-dioxygenases, there was no spectral evidence for the presence of a chromophoric cofactor. EPR analyses of MeQDO did not reveal any signal that could be assigned to an organic radical species or to a metal, and X-ray fluorescence spectrometry of both enzymes did not show any metal present in stoichiometric amounts. Ethylxanthate, metal-chelating agents (tiron, alpha, alpha'-bipyridyl, 8-hydroxyquinoline, o-phenanthroline, EDTA, diphenylthiocarbazone, diethyldithiocarbamate), reagents that modify sulfhydryl groups (iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate), and reducing agents (sodium dithionite, dithiothreitol, mercaptoethanol) either did not affect 2,4-dioxygenolytic activities at all or inhibited at high concentrations only. With respect to the supposed lack of any cofactor and with respect to the inhibitors of dioxygenolytic activities, MeQDO and QDO resemble aci-reductone oxidase (CO-forming) from Klebsiella pneumoniae, which catalyzes 1,3-dioxygenolytic cleavage of 1,2-dihydroxy-3-keto-S-methylthiopentene anion (Wray, J. W. & Abeles, R. H. (1993) J. Biol. Chem. 268, 21466-21469; Wray, J. W. & Abeles, R. H. (1995) J. Biol. Chem. 270, 3147-3153). 1H-3-Hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline were reactive towards molecular oxygen in the presence of the base catalyst potassium-tert.-butoxide in the aprotic solvent N,N-dimethylformamide. Base-catalyzed oxidation, yielding the same products as the enzyme-catalyzed conversions, provides a non-enzymic model reaction for 2,4-dioxygenolytic release of CO from 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline.

Cloning, expression, and sequence analysis of the three genes encoding quinoline 2-oxidoreductase, a molybdenum-containing hydroxylase from Pseudomonas putida 86.

The three genes coding for quinoline 2-oxidoreductase (Qor) of Pseudomonas putida 86 were cloned and sequenced. The qor genes are clustered in the transcriptional order medium (M) small (S), large (L) and code for three subunits of 288 (QorM), 168 (QorS), and 788 (QorL) amino acids, respectively. Formation of active quinoline 2-oxidoreductase and degradation of quinoline occurred in a recombinant P. putida KT2440 clone. The amino acid sequences of Qor show significant homology to various prokaryotic molybdenum containing hydroxylases and to eukaryotic xanthine dehydrogenases. QorS contains two conserved motifs for [2Fe-2S] clusters. The binding motif for the N-terminal [2Fe-2S] cluster corresponds to the binding site of bacterial and chloroplast-type [2Fe-2S] ferredoxins, whereas the amino acid pattern of the internal [2Fe-2S] center apparently is a distinct feature of molybdenum-containing hydroxylases, showing no homology to any other described [2Fe-2S] binding motif. The medium subunit QorM presumably contains the FAD, but no conserved sequence areas or described motifs of FAD, NAD, NADP, or ATP binding were detected. Putative binding sites of the molybdopterin cytosine dinucleotide cofactor were detected in QorL by comparison with "contacting segments" recently described in aldehyde oxidoreductase from Desulfovibrio gigas (Romão, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170-1176).

Hydroxylation of quinaldic acid: quinaldic acid 4-monooxygenase from Alcaligenes sp. F-2 versus quinaldic acid 4-oxidoreductases.

The N-heterocycles quinaldic acid (quinoline 2-carboxylic acid), kynurenic acid (4-hydroxyquinoline 2-carboxylic acid), 2-oxo-1,2-dihydroquinoline, and xanthine are utilized by Alcaligenes sp. F-2 as sole source of carbon and energy. Although quinoline did not serve as growth substrate, 8-hydroxy-2-oxo-1,2-dihydroquinoline and 8-hydroxycoumarin, metabolites of the 'coumarin pathway' of quinoline catabolism, were isolated from the culture fluid during growth on 2-oxo-1,2-dihydroquinoline. Contrary to Serratia marcescens 2CC-1 and Pseudomonas sp. AK-2 (Sauter et al. (1993) Biol. Chem. Hoppe-Seyler 374, 1037-1046), which possess different molybdenum-containing hydroxylases catalysing the 4-hydroxylation of quinaldic acid to kynurenic acid with incorporation of oxygen derived from water and concomitant reduction of an electron acceptor, Alcaligenes sp. F-2 contains an inducible quinaldic acid 4-monooxygenase that catalyses the very same conversion in the presence of O2 and NADH. The activity of the monooxygenase was enhanced 1.5-fold by Fe2+ ions. The extremely thermolabile enzyme (apparent molecular mass: 155 kDa) exclusively accepted quinaldic acid as substrate. The 'pseudosubstrates' menadione, 8-hydroxyquinoline, and 8-hydroxy-2-oxo-1,2-dihydroquinoline effected consumption of NADH and oxygen without being hydroxylated. Quinaldic acid 4-monooxygenase was inhibited by sulfhydryl modifying and chelating agents, and by various divalent metal ions, whereas reducing agents did not affect enzymatic activity.

Quinaldine 4-oxidase from Arthrobacter sp. Rü61a, a versatile procaryotic molybdenum-containing hydroxylase active towards N-containing heterocyclic compounds and aromatic aldehydes.

Quinaldine 4-oxidase from Arthrobacter sp. Rü61a, an inducible molybdenum-containing hydroxylase, was purified to homogeneity by an optimized five-step procedure. Molecular oxygen is proposed as physiological electron acceptor. Electrons are also transferred to artificial electron acceptors with E'o > -8 mV. The molybdo-iron/sulfur flavoprotein regiospecifically attacks its N-heterocyclic substrates: isoquinoline and phthalazine are hydroxylated adjacent to the N-heteroatom at Cl, whereas quinaldine, quinoline, cinnoline and quinazoline are hydroxylated at C4. Additionally, the aromatic aldehydes benzaldehyde, salicylaldehyde, vanillin and cinnamaldehyde are oxidized to the corresponding carboxylic acids, whereas short-chain aliphatic aldehydes are not. Quinaldine 4-oxidase is compared to the two molybdenum-containing hydroxylases quinoline 2-oxidoreductase from Pseudomonas putida 86 [Tshisuaka, B., Kappl, R., Hüttermann, J. & Lingens, F. (1993) Biochemistry 32, 12928-12934] and isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7 [Lehmann, M., Tshisuaka, B., Fetzner, S., Röger, P. & Lingens, F. (1994) J. Biol. Chem. 269, 11254-11260] with respect to the substrates converted and the electron-acceptor specificities. These dehydrogenases hydroxylate their N-heterocyclic substrates exclusively adjacent to the heteroatom. Whereas the aldehydes tested are scarcely oxidized by quinoline 2-oxidoreductase, isoquinoline 1-oxidoreductase catalyzes the oxidation of the aromatic aldehydes, although being progressively inhibited. Neither quinoline 2-oxidoreductase nor isoquinoline 1-oxidoreductase transfer electrons to oxygen. Otherwise, the spectrum of electron acceptors used by quinoline 2-oxidoreductase and quinaldine 4-oxidase is identical. However, isoquinoline 1-oxidoreductase differs in its electron-acceptor specificity. Quinaldine 4-oxidase is unusual in its substrate and electron-acceptor specificity. This enzyme is able to function as oxidase or dehydrogenase, it oxidizes aldehydes, and it catalyzes the nucleophilic attack of N-containing heterocyclic compounds at two varying positions depending on the substrate.

EPR, electron spin echo envelope modulation, and electron nuclear double resonance studies of the 2Fe2S centers of the 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS.

The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F. (1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz = 2.025, gy = 1.91, gx = 1.79, gav = 1.91, Em = -125 +/- 10 mV; ferredoxin-type center with gz = 2.05, gy = 1.96, gx = 1.89, gav = 1.97, Em = -200 +/- 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em = +320 mV) and the water-soluble ferredoxin (Em = -430 mV) of spinach chloroplasts, as typical representatives of the gav = 1.91 and gav = 1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed.

The 2Fe2S centres of the 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy.

The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity. We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system. The oxygenase component was identified as a Rieske [2Fe2S] protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gav = 1.893. The reductase component, an iron-sulfur flavoprotein, contained a [2Fe2S] cluster with gz,y,x = 2.03, 1.94, 1.89 and the average g-value (gav) of 1.953, typical of a ferredoxin-type centre. In redox titrations at pH 7, the midpoint potentials were determined to be -180 mV +/- 30 mV and -100 mV +/- 10 mV for the reductase and oxygenase component, respectively. A detailed comparison to other multicomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved.

Quinoline 2-oxidoreductase and 2-oxo-1,2-dihydroquinoline 5,6-dioxygenase from Comamonas testosteroni 63. The first two enzymes in quinoline and 3-methylquinoline degradation.

The enzymes catalysing the first two steps of quinoline and 3-methylquinoline degradation by Comamonas testosteroni 63 were investigated. Quinoline 2-oxidoreductase, which catalyses the hydroxylation of (3-methyl-)quinoline to (3-methyl-)2-oxo-1,2-dihydroquinoline, was purified to apparent homogeneity. The native enzyme, with a molecular mass of 360 kDa, is composed of three non-identical subunits (87, 32, and 22 kDa), occurring in a ratio of 1.16:1:0.83. Containing FAD, molybdenum, iron, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8, the enzyme belongs to the molybdo-iron/sulfur flavoproteins. Molybdopterin cytosine dinucleotide is the organic part of the pterin molybdenum cofactor. Comparison of N-terminal amino acid sequences revealed similarities to a number of procaryotic molybdenum-containing hydroxylases. Especially the N-termini of the beta-subunits of the quinoline 2-oxidoreductases from Comamonas testosteroni 63, Pseudomonas putida 86, and Rhodococcus spec. B1, and of quinoline-4-carboxylic acid 2-oxidoreductase from Agrobacterium spec. 1B showed striking similarities. Further degradation of (3-methyl-)2-oxo-1,2-dihydroquinoline proceeds via dioxygenation at the benzene ring, i.e. at 5,6-position [Schach, S., Schwarz, G., Fetzner, S. & Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 175-181]. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase was partially purified; NADH and oxygen are required for the reaction, and the enzymic activity is enhanced 1.5-fold by addition of Fe2+ ions. Unexpectedly, this aromatic ring dioxygenase did not separate into distinct protein components, but is apparently a single-component enzyme. The molecular mass was estimated to be about 260 kDa. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is very thermolabile. However, dithioerythritol and low concentrations of substrate had a moderately stabilizing effect. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is inhibited by sulfhydryl-blocking agents, by metal-chelating agents, and by the flavin analogues quinacrine and acriflavin.

2-Oxo-1,2-dihydroquinoline 8-monooxygenase, a two-component enzyme system from Pseudomonas putida 86.

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 (M(r), 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 (M(r), 330,000) consists of six identical subunits (M(r), 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.