Gene cluster of Arthrobacter ilicis Ru61a involved in the degradation of quinaldine to anthranilate: characterization and functional expression of the quinaldine 4-oxidase qoxLMS genes.
A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü61a 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.
AG Mikrobiologie, Institut für Chemie und Biologie des Meeres, Carl von Ossietzky Universität Oldenburg, D-26111 Oldenburg, Germany.
Katja
K
Parschat
Bernhard
B
Hauer
Reinhard
R
Kappl
Roswitha
R
Kraft
Jurgen
J
Huttermann
Susanne
S
Fetzner
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D001173:Arthrobacter / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D016958:Pseudomonas putida / Q000378:metabolism; D011798:Quinaldines / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D008667:Metalloproteins; D011798:Quinaldines; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D008982:Molybdenum; D004247:DNA; C037073:2-methylquinoline; D010088:Oxidoreductases; C080754:quinaldine oxidoreductase; D000604:Amino Acyl-tRNA Synthetases