Purification and characterisation of the enantiospecific dioxygenases from Delftia acidovorans MCI initiating the degradation of phenoxypropionate and phenoxyacetate herbicides

摘要

After cultivation on (R,S)-2-(2,4-dichlorophenoxy)propionate, two a-ketoglutarate-dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R-enantiomer of substituted phenoxypropionate derivatives: the K-m values were 55 muM and 30 muM, the k(cat) values 55 min(-1) and 34 min(-1) with (R)-2-(2,4-dichlorophenoxy)propionate [(R)-2,4-DP] and (R)-2-(4-chloro-2-methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S-enantiomers with K-m values of 49 muM and 22 muM, and keat values of 50 min(-1) and 46 min(-1) with (S)-2-(2,4-dichlorophenoxy)propionate [(S)-2,4-DP] and (S)-2-(4-chloro2-methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4-dichlorophenoxyacetate: K-m = 123 muM, k(cat) = 36 min(-1)) with significant activity. As the second substrate, only a-ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K-i values of 3 mM with (R)-2,4-DP and 1.5 mM with (S)-2,4-DP. The reaction was strictly dependent on the presence of Fe2+ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 muM diethylpyrocarbonate (DEPC).