Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene from Arthrobacter spec. Rü61a.

The ring cleaving enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD)) of Arthrobacter spec. Rü61a is part of the quinaldine degradation pathway. Carbon monoxide and N-acetyl-anthranilate are the products formed by dioxygenolytic cleavage of two C-C bonds in the substrate's pyridine ring. The gene coding for HOD was cloned and sequenced. An isoelectric point of pH 5.40 and a molecular mass of 31,838 Da was deduced from the sequence. HOD is shown to be remarkably similar to 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) of Pseudomonas putida 33/1, but not to other dioxygenases described so far. Consensus regions indicative for any chromophoric cofactor or any catalytically relevant metal were not detected. Sequence comparisons and secondary structure predictions revealed HOD as a new member of the alpha/beta hydrolase fold family. Expression in E. coli yielded recombinant catalytically active His-tagged HOD. S101A and D233A, two mutants of HOD, were obtained by site-directed mutagenesis. Since their residual activity is 43.1% and 62.6%, respectively, they probably are of no catalytic relevance although they might play a role in the interaction between enzyme and substrate.

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.

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).

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.

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1.

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.

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).

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3. Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity.

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45 degrees C. Chloride ions were inhibitory, and competitive with respect to NADH. 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-hydroxybenzoate, 2-chloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed. The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

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.

Dehalogenation of 4-chlorobenzoate. Characterisation of 4-chlorobenzoyl-coenzyme A dehalogenase from Pseudomonas sp. CBS3.

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg2+ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 degrees C. The apparent Km value for 4-chlorobenzoyl-coenzyme A was 2.4-2.7 microM. Vmax was 1.1 x 10(-7) M sec-1 (2.2 mumol min-1 mg-1 of protein). The NH2-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.

Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1.

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.

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.

Molecular cloning of the isoquinoline 1-oxidoreductase genes from Pseudomonas diminuta 7, structural analysis of iorA and iorB, and sequence comparisons with other molybdenum-containing hydroxylases.

The iorA and iorB genes from the isoquinoline-degrading bacterium Pseudomonas diminuta 7, encoding the heterodimeric molybdo-iron-sulfur-protein isoquinoline 1-oxidoreductase, were cloned and sequenced. The deduced amino acid sequences IorA and IorB showed homologies (i) to the small (gamma) and large (alpha) subunits of complex molybdenum-containing hydroxylases (alpha beta gamma/alpha 2 beta 2 gamma 2) possessing a pterin molybdenum cofactor with a monooxo-monosulfido-type molybdenum center, (ii) to the N- and C-terminal regions of aldehyde oxidoreductase from Desulfovibrio gigas, and (iii) to the N- and C-terminal domains of eucaryotic xanthine dehydrogenases, respectively. The closest similarity to IorB was shown by aldehyde dehydrogenase (Adh) from the acetic acid bacterium Acetobacter polyoxogenes. Five conserved domains of IorB were identified by multiple sequence alignments. Whereas IorB and Adh showed an identical sequential arrangement of these conserved domains, in all other molybdenum-containing hydroxylases the relative position of "domain A" differed. IorA contained eight conserved cysteine residues. The amino acid pattern harboring the four cysteine residues proposed to ligate the Fe/S I cluster was homologous to the consensus binding site of bacterial and chloroplast-type [2Fe-2S] ferredoxins, whereas the pattern including the four cysteines assumed to ligate the Fe/S II center showed no similarities to any described [2Fe-2S] binding motif. The N-terminal region of IorB comprised a putative signal peptide similar to typical leader peptides, indicating that isoquinoline 1-oxidoreductase is associated with the cell membrane.

Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS.

The two-component nonheme iron dioxygenase system 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS catalyzes the double hydroxylation of 2-halobenzoates with concomitant release of halogenide and carbon dioxide, yielding catechol. The gene cluster encoding this enzyme, cbdABC, was localized on a 70-kbp conjugative plasmid designated pBAH1. The nucleotide sequences of cbdABC and flanking regions were determined. In the deduced amino acid sequence of the large subunit of the terminal oxygenase component (CbdA), a conserved motif proposed to bind the Rieske-type [2Fe-2S] cluster was identified. In the NADH:acceptor reductase component (CbdC), a putative binding site for a chloroplast-type [2Fe-2S] center and possible flavin adenine dinucleotide- and NAD-binding domains were identified. The cbdABC sequences show significant homology to benABC, which encode benzoate 1,2-dioxygenase from Acinetobacter calcoaceticus (52% identity at the deduced amino acid level), and to xylXYZ, which encode toluate 1,2-dioxygenase from Pseudomonas putida mt-2 (51% amino acid identity). Recombinant pkT231 harboring cbdABC and flanking regions complemented a plasmid-free mutant of wild-type P. cepacia 2CBS for growth on 2-chlorobenzoate, and it also allowed recombinant P. putida KT2440 to metabolize 2-chlorobenzoate. Functional NADH:acceptor reductase and oxygenase components of 2-halobenzoate 1,2-dioxygenase were enriched from recombinant Pseudomonas clones.

Purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase from Streptomyces rochei 303: comparison with an analogous enzyme from Azotobacter sp. strain GP1.

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiol-cleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (kcat/Km = 1.2 and 0.57 s-1.microM-1, respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH2-terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.