CLONING OF GENES INVOLVED IN CARBAZOLE DEGRADATION OF PSEUDOMONAS SP STRAIN CA10 - NUCLEOTIDE-SEQUENCES OF GENES AND CHARACTERIZATION OF META-CLEAVAGE ENZYMES AND HYDROLASE

The DNA fragment encoding meta cleavage enzymes and the meta-cleavage compound hydrolase, involved in carbazole degradation, was cloned from the carbazole-utilizing bacterium Pseudomonas sp. strain CA10. DNA se quence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene ord er). ORF1 and ORF2 were indispensable for meta-cleavage activity for 2 '-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydrox ybiphenyl, and were designated carBa and carBb, respectively. The alig nment of CarBb with other meta-cleavage enzymes indicated that CarBb m ay have a non heme iron cofactor coordinating site, On the basis of th e phylogenetic tree, CarBb was classified as a member of the protocate chuate 4,5-dioxygenase family. This unique extradiol dioxygenase, CarB , had significantly higher affinity and about 20-times-higher meta-cle avage activity for 2,3-dihydroxybiphenyl than for catechol derivatives . The putative polypeptide encoded by ORF3 was homologous,vith meta-cl eavage compound hydrolases in other bacteria, and ORF3 was designated carC. The hydrolase activity of CarC for 2-hydroxy-6-oxo-6-phenylhexa- 2,4-dienoic acid, the meta-cleavage compound of 2,3-dihydroxybiphenyl, was 40 times higher than that for 2-hydroxy-6-oxohepta-2,4-dienoic ac id, the meta-cleavage compound of 3-methylcatechol. Alignment analysis and the phylogenetic tree indicate that CarC has greatest homologies with hydrolases involved in the monoaromatic compound degradation path way, These results suggest the possibility that CarC is a novel type o f hydrolase.