Poh_2001_Microbios_105_43

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 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-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. 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 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.