Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobacter radioresistens

Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been pur ified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homo dimers composed of a single type of subunit with molecular mass of 38,600 a nd 37,700, Da respectively. In conditions of low ionic strength, IsoA can a ggregate as a trimer, in contrast to IsoB, which maintains the dimeric stru cture, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown o n phenol, whereas the IsoB is selectively expressed using benzoate as carbo n source. This is the first evidence of the presence of differently express ed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB cont ain approximately 1 iron(III) ion per subunit and both show electronic abso rbance and EPR features typical of Fe(III) intradiol dioxygenases. The kine tic properties of the two enzymes such as the specificities toward substitu ted catechols, the main catalytic parameters, and their behavior in the pre sence of different kind of inhibitors are, unexpectedly, very similar, in c ontrast to most of the previously known dioxygenase isozymes.