Cloning, Expression, and Sequence Analysis of the Three Genes Encoding Quinoline 2-Oxidoreductase, a Molybdenum-containing Hydroxylase from   86

Bläse, Marcel; Bruntner, Christina; Tshisuaka, Barbara; Fetzner, Susanne; Lingens, Franz

doi:10.1074/jbc.271.38.23068

Cited by 39 publications

(48 citation statements)

References 52 publications

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“…In contrast, an E. coli HB101 pVK55B/5 clone did not transform quinaldine, and crude extracts did not show Qox activity. This observation is in accordance with other reports on futile attempts to express genes coding for MCD-containing hydroxylases in E. coli (27,28).…”

Section: Degradative Capacities Of P Putida Kt2440 Cosmid Clones Andsupporting

confidence: 93%

“…manipulation and for the regulated, functional expression of the genes coding for the enzyme to be investigated. Whereas genes coding for molybdenum hydroxylases containing molybdopterin or the molybdopterin guanine dinucleotide form of the cofactor have been successfully expressed in Escherichia coli (24 -26), attempts to produce MCD-containing enzymes in E. coli clones failed (27,28). 2 We have recently been working at the construction of expression clones for the synthesis of Qor and Ior.…”

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confidence: 99%

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Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

Parschat

Hauer

Kappl

et al. 2003

Journal of Biological Chemistry

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A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü 61a was cloned into the cosmid pVK100. Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate. The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase. The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1. Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P. putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P. putida KT2440 pKP1 were essentially like those of wild-type Qox. This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis. Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located. Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport.The genetic diversity and flexibility of prokaryotes has led to the evolution of an impressive variety of metabolic pathways to transform or degrade natural as well as numerous xenobiotic compounds. The genes coding for enzymes involved in degradative pathways are often organized as operons and supraoperonic clusters comprising "pathway modules" (1-4).N-Heteroaromatic compounds are metabolized and even mineralized by various bacteria (for a review, see Ref. 5 and references therein). Quinaldine (2-methylquinoline) is utilized by Arthrobacter ilicis Rü 61a as a source of carbon, nitrogen, and energy; its degradation proceeds via the "anthranilate pathway" (5, 6). The initial step, the hydroxylation of quinaldine in para position to the N-heteroatom, is catalyzed by the inducible enzyme quinaldine 4-oxidase (Qox). 1 Qox is a molybdo-iron/sulfur-flavoprotein with an (LMS) 2 subunit structure and has been classified to belong to the xanthine oxidase family of molybdenum enzymes (7-10; for reviews on molybdenum enzymes, see Refs. 11-13). Like many other bacterial molybdenum hydroxylases, e.g. quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 (14, 15), isoquinoline 1-oxidoreductase (Ior...

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Section: Degradative Capacities Of P Putida Kt2440 Cosmid Clones Andsupporting

confidence: 93%

mentioning

confidence: 99%

Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

Parschat

Hauer

Kappl

et al. 2003

Journal of Biological Chemistry

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“…We therefore suggest that PucD consists of the subunit of B. subtilis xanthine dehydrogenase that binds the substrate xanthine and the molybdenum cofactor. pucE and pucC both encode polypeptides with amino acid sequence identity to subunits of different dehydrogenases (2,15,40). The function of these subunits is to bind essential prosthetic groups like [2Fe-2S] clusters and flavin adenine dinucleotide (FAD) involved in the oxidation reaction catalyzed by the dehydrogenase holoenzymes.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

2001

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The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285°on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3-to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

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“…The recombinant clone P. putida mt-2 KT2440 (13/42), harboring the cosmid vector pCIB119 with a 30-kb insertion of genomic DNA of P. putida 86, was reported to carry and express the genes for quinoline 2-oxidoreductase, the first enzyme of the quinoline degradation pathway in P. putida 86 (2). In this study, the expression of 2-oxo-1,2-dihydroquinoline 8-monooxygenase, the second enzyme in this degradation pathway, was investigated, using the same recombinant strain.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1997

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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

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Cloning, Expression, and Sequence Analysis of the Three Genes Encoding Quinoline 2-Oxidoreductase, a Molybdenum-containing Hydroxylase from 86

Cited by 39 publications

References 52 publications

Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

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

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