PURIFICATION, CHARACTERIZATION, AND GENE ANALYSIS OF CATECHOL 2,3-DIOXYGENASE FROM THE ANILINE-ASSIMILATING BACTERIUM PSEUDOMONAS SPECIES AW-2

Catechol 2,3-dioxygenase (C23D; EC 1.13.1.2) was purified to homogenei ty from a cell extract of Pseudomonas sp. AW-2 grown on aniline, and t he purified C23D was characterized. The molecular mass estimated by ge l filtration was 110 kDa. The enzyme dissociated into four identical s ubunits each with the molecular mass of 33 kDa. The enzyme had high ac tivity for 3-methylcatechol as well as catechol, and differed from the enzyme from Pseudomonas putida mt-2, which carries the TOL plasmid, i n optimal on for catechol, extradiol cleavage activities for 3-methylc atechol and 4-methylcatechol, and immunochemical properties. The amino acid sequence deduced from a C23D gene, alnE, from Pseudomonas sp. AW -2 was 85.7% identical to that of 3-methylcatechol 2,3-dioxygenase fro m toluidine-assimilating Pseudomonas putida UCC22. AlnE was 44.1% iden tical to the C23D encoded by xylE in P. putida mt-2. Because XylE has low activity for 3-methylcatechol, these results suggest that the diff erences in substrate specificity for 3-methylcatechol among the C23Ds reflected their sequence similarity.