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.
Parschat
Katja
K
AG Mikrobiologie, Institut für Chemie und Biologie des Meeres, Carl von Ossietzky Universität Oldenburg, D-26111 Oldenburg, Germany.
Hauer
Bernhard
B
Kappl
Reinhard
R
Kraft
Roswitha
R
Huttermann
Jurgen
J
Fetzner
Susanne
S
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