Abstract
The tfdC(I)D(I)E(I)F(I,) and tfdD(II)C(II)E(II)F(II) gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd(I)-encoded enzymes was usually higher than that of tfd(II)-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD(II)-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.