2,4-Dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134

2,4-Dichlorophenoxyacetate (2,4-D)/alpha -ketoglutarate (alpha -KG) dioxyge nase has been purified to apparent homogeneity from Burkholderia cepacia st rain 2a, which utilizes 2.4-D as sole carbon source. The enzyme required fe rrous ions, and was a homodimer composed of subunits having an M-r of simil ar to 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha -KG and dioxygen, with the production of one mol each of succinate, 2,4-dic hlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degreesC, and reactivity was enhanced by the presence of ascorbate and cys teine. Mn2+, Zn2+, Cu2+ Fe3+ and Co2+ were inhibitory, and chemical modific ation of the dioxygenase revealed that thiol groups were essential for acti vity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha -KG were 109 and 8.9 muM, respectively. The properties of this e nzyme are compared with those of the 2,4-D/alpha -KG dioxygenase from Ralst onia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequ ence, and a different temperature 'optimum', pH optimum, substrate specific ity and sensitivity to thiol-binding reagents.