Importance of different tfd genes for degradation of chloroaromatics by Ralstonia eutropha JMP134

The tfdC_ID_IE_IF_I, and tfdD_IIC_IIE_IIF_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.