CLONING, EXPRESSION, AND SEQUENCE-ANALYSIS OF THE 3 GENES ENCODING QUINOLINE 2-OXIDOREDUCTASE, A MOLYBDENUM-CONTAINING HYDROXYLASE FROM PSEUDOMONAS-PUTIDA-86

The three genes coding for quinoline a-oxidoreductase (Qor) of Pseudom onas 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 aci ds, respectively. Formation of active quinoline 2-oxidoreductase and d egradation of quinoline occurred in a recombinant P. putida KT2440 clo ne. The amino acid sequences of Qor show significant homology to vario us prokaryotic molybdenum containing hydroxylases and to eukaryotic xa nthine dehydrogenases. QorS contains two conserved motifs for [2Fe-2S] clusters. The binding motif for the N-terminal [2Fe-2S] cluster corre sponds to the binding site of bacterial and chloroplast-type [2Fe-2S] ferredoxins, whereas the amino acid pattern of the internal [2Fe-2S] c enter apparently is a distinct feature of molybdenum-containing hydrox ylases, showing no homology to any other described [2Fe-2S] binding mo tif. The medium subunit QorM presumably contains the FAD, but no conse rved sequence areas or described motifs of FAD, NAD, NADP, or ATP bind ing were detected. Putative binding sites of the molybdopterin cytosin e dinucleotide cofactor were detected in QorL by comparison with ''con tacting segments'' recently described in aldehyde oxidoreductase from Desulfovibrio gigas (Romao, 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).