2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134.
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.
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School of Chemical and Life Sciences, University of Greenwich, London, Great Britain.
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Bruce
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Smith
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D015084:2,4-Dichlorophenoxyacetic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics; D002614:Chelating Agents / Q000494:pharmacology; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D005296:Ferrous Compounds / Q000378:metabolism; D006540:Herbicides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D002614:Chelating Agents; D005296:Ferrous Compounds; D006540:Herbicides; D008670:Metals; D013438:Sulfhydryl Compounds; D015084:2,4-Dichlorophenoxyacetic Acid; D006899:Mixed Function Oxygenases; C084087:2,4-dichlorophenoxyacetate-alpha-ketoglutarate dioxygenase