Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from Pseudomonas aeruginosa ZD 4-3

Catechol 2,3-dioxygenase (C23O), an extradiol-type dioxygenase cleaving the aromatic C—C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxy-muconic semialdehyde. Based on a curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from Pseudomonas aeruginosa ZD 4-3, which was able to degrade both single and bicyclic compounds via a meta-cleavage path-way. A complete nucleotide sequence analysis of the C23O revealed that it has one ORF, which showed a strong overall amino acid similarity to the known gram-negative bacterial mesophilic C23Os. The alignment analysis indicated a distinct difference between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases that cleave bicyclic aromatic compounds. The heterogeneous expression of the pheB gene in E. Coli BL21(DE3) demonstrated that this C23O possesses a meta-cleavage activity.