Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31

Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that c onverts 3-chlorocatechol and 3-methylcatechol, which enables the organism t o use both chloroaromatics and methylaromatics for growth, A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2 ,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeased to b e plasmid localized and was found in a region that also harbors genes encod ing a transposase, a ferredoxin that was homologous to XyIT, an open readin g frame with similarity to a protein of a meta-cleavage pathway with unknow n function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was mos t similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extra diol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930-5937 , 1996). The substrate range and turnover capacity with 3-chlorocatechol we re determined for CbzE and four related catechol 2,3-dioxygenases. The resu lts showed that CbzE was the only enzyme that could productively convert 3- chlorocatechol, Besides, CbzE was less susceptible to inactivation by methy lated catechols, Hybrid enzymes that were made of CzbE and the catechol 2,3 -dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.