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

Abstract

Multicomponent oxygenases play an important role in the bacterial degradation of aromatic compounds. 2-Oxo-1,2-dihydroquinoline 8-monooxygenase catalyzes the second step of quinoline degradation by Pseudomonas putida 86: in a NADHdependent oxygenation, 2-oxo-1,2-dihydroquinoline is converted to 8-hydroxy-2-oxo-1,2-dihydroquinoline (Fig. 1). As illustrated in Fig. 1, this enzyme system consists of two soluble protein components with four redox active centers, which constitute an electron transfer chain. Electrons are transferred from NADH via flavin adenine dinucleotide (FAD) and a chloroplast-type [2Fe-2S] cluster, which are located on the reductase component, to the substrate hydroxylating oxygenase component, which harbors Rieske-type [2Fe-2S] clusters and additional iron (26,27).Based on the number of its protein components and on its set of cofactors, the enzyme system belongs to the class IB multicomponent Rieske center nonheme iron oxygenases as defined by Batie et al. (1). However, it differs from known class IB enzymes, since the oxygenase component is a homomultimer and thus resembles class IA oxygenase components (27). This unusual property prompted us to investigate the phylogenetic relationship of multicomponent oxygenases.Here we report the localization, expression, and comparative sequence analysis of the oxoO and oxoR genes, encoding the oxygenase and reductase components of 2-oxo-1,2-dihydroquinoline 8-monooxygenase from P. putida 86. Putative cofactor binding domains are located, and based on sequence alignments, the phylogenetic relationship of multicomponent oxygenases is discussed. MATERIALS AND METHODSBacterial strains and plasmids. P. putida 86 had been isolated from soil by selective enrichment on quinoline as the carbon source (29). The recombinant strain P. putida mt-2 KT2440 (13/42) harbors the cosmid pCIB119 with a 30-kb insertion of genomic DNA of P. putida 86 and has been described previously (2). Fragments of this insert were cloned in the vector plasmid pUC18 (23, 37) with Escherichia coli TG1 (8) as the host strain. The plasmid pCIB119 is a double cosmid and was a kind gift of Stephen T. Lam (Ciba-Geigy, Research Triangle Park, N.C.).Media and growth conditions. For the preparation of plasmid DNA, overnight cultures in Luria-Bertani medium (28) with tetracycline, 50 g/ml (for pCIB119), or ampicillin, 100 g/ml (for pUC18), were used. Cometabolic conversion of 2-oxo-1,2-dihydroquinoline was investigated by using mineral salt medium (7) with 2-g/liter succinate and 40-mg/liter 2-oxo-1,2-dihydroquinoline. Pseudomonas strains were grown at 30°C, and E. coli strains were grown at 37°C. DNA techniques. Plasmid DNA was prepared by alkaline lysis (28) or by using the Midi kit (Qiagen, Inc., Chatsworth, Calif.). DNA fragments were isolated from agarose gels according to the instruction manual for the Qiaex II gel extraction kit (Qiagen). Restriction digestions, dephosphorylation of DNA fragments, and DNA ligation were performed as described by the enzyme suppliers

Catabolism of Phenylacetic Acid in Escherichia coli

“…These enzymes are members of the ferredoxin-NADP ϩ reductase (FNR) family and they contain a FNR-like domain consisting of a FMN(FAD)-and a NAD(P)-binding region (57). The residues 55 RCYS 58 in PaaE fit the RXYS consensus motif for binding of the isoalloxazine ring of the flavin cofactor, and residues 121 GS-GITP 126 and 216 CGPAAM 221 match the GXG(X) 2-3 P and CG(X) [3][4] M sequences for the binding of the NAD(P) ribose and NAD(P)-pyrophosphate-nicotinamide moieties of the nicotinamide cofactor, respectively (58). At the C terminus of the FNR-like domain, residues 299 -337 in PaaE correspond to the CX 4 CXXCX 24 -34 C conserved motif of the plant-type ferredoxin [2Fe-2S] binding domain (58).…”

Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

“…Our finding in the present study that FAD acts as a KshB cofactor thus is not consistent with the classification of this protein in class IA. Several other examples of enzymes that do not fit the Batie classification of ring-hydroxylating oxygenases have been reported (19,31,33). Another classification system has been proposed for oxygenase components involved in ring-hydroxylating oxygenations (28).…”

A Three-component Dicamba O-Demethylase from Pseudomonas maltophilia, Strain DI-6

“…ADP1. Both of these homologous enzymes are two-component monooxygenase systems in which electrons are shuttled from the low-potential donor NADH to oxygen via a reductase component that contains flavin (FAD or FMN) and NAD binding sites and a [2Fe-2S] cluster (21). In contrast, dicamba O-demethylase is a three-component enzyme system in which the [2Fe-2S] cluster that is involved in the transfer of electrons from NADH to oxygen is not contained within the reductase DIC component, but is associated with a separate ferredoxin DIC molecule.…”