CHLOROCATECHOL 1,2-DIOXYGENASE FROM RHODOCOCCUS-ERYTHROPOLIS 1CP - KINETIC AND IMMUNOCHEMICAL COMPARISON WITH ANALOGOUS ENZYMES FROM GRAM-NEGATIVE STRAINS

Chlorocatechol 1,2-dioxygenase from Rhodococcus erq,thropolis 1CP was purified to homogeneity. In contrast to chlorocatechol 1,2-dioxygenase from Gram-negative strains which have a very broad substrate toleranc e, the Rhodococcus enzyme was relatively more specific and had a disti nct preference for 4-substituted catechols. Protein and metal analysis indicate an unusual stoichiometry of one atom each of iron and mangan ese/64-kDa homodimer. The N-terminal amino acid sequence (27 residues) of the Rhodococcus chlorocatechol 1,2-dioxygenase was determined and exhibited 15-22% identity to the published sequences of catechol 1,2-d ioxygenases and other chlorocatechol 1,2-dioxygenases. Antiserum was r aised in rabbits and antibodies against Rhodococcus chlorocatechol 1,2 -dioxygenase were affinity purified. Dot-blot analysis revealed a very weak reaction between the antibodies and partially purified chlorocat echol 1,2-dioxygenases from Alcaligenes eutrophus JMP134 and Pseudomon as putida 87. No reaction between these antibodies and above enzymes w as observed using Western blotting. Kinetic and immunochemical data as well as comparison of subunit molecular mass and suggest that the Rho dococcus enzyme differs significantly from the known highly similar ch lorocatechol 1,2-dioxygenases of Gram-negative strains and seems to be only distantly related to them.