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 sequence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene order). ORF1 and ORF2 were indispensable for meta-cleavage activity for 2'-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydroxybiphenyl, and were designated carBa and carBb, respectively. The alignment of CarBb with other meta-cleavage enzymes indicated that CarBb may have a non-heme iron cofactor coordinating site. On the basis of the phylogenetic tree, CarBb was classified as a member of the protocatechuate 4,5-dioxygenase family. This unique extradiol dioxygenase, CarB, had significantly higher affinity and about 20-times-higher meta-cleavage activity for 2,3-dihydroxybiphenyl than for catechol derivatives. The putative polypeptide encoded by ORF3 was homologous with meta-cleavage 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 acid, 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 pathway. These results suggest the possibility that CarC is a novel type of hydrolase.
Sato
S I
SI
Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Japan.
Ouchiyama
N
N
Kimura
T
T
Nojiri
H
H
Yamane
H
H
Omori
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002227:Carbazoles / Q000378:metabolism*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D049308:Dioxygenases; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006867:Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010105:Oxygenases / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D002227:Carbazoles; D004269:DNA, Bacterial; C041514:carbazole; D010105:Oxygenases; D049308:Dioxygenases; C022476:protocatechuate 4,5-dioxygenase; D006867:Hydrolases