Open AccessPurification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides
Author(s) -
Westendorf A.,
Müller R. H.,
Babel W.
Publication year - 2003
Publication title -
acta biotechnologica
Language(s) - English
Resource type - Journals
eISSN - 1521-3846
pISSN - 0138-4988
DOI - 10.1002/abio.200390006
Subject(s) - propionate , chemistry , stereochemistry , enzyme , substrate (aquarium) , enantiomer , bond cleavage , kinetics , enzyme kinetics , ether , divalent , medicinal chemistry , biochemistry , active site , catalysis , organic chemistry , biology , ecology , physics , quantum mechanics
After cultivation on ( R,S )‐2‐(2,4‐dichlorophenoxy)propionate, two α ‐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 μM and 30 μM, 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 μM and 22 μM, and k cat values of 50 min –1 and 46 min –1 with ( S )‐2‐(2,4‐dichlorophenoxy)propionate [( S )‐2,4‐DP] and ( S )‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K m = 123 μM, k cat = 36 min –1 ) with significant activity. As the second substrate, only α ‐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 Fe 2+ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).