SummaryAfter 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).
The rdpA gene of strains Delftia acidovorans MC1, Rhodoferax sp. P230, and Sphingobium herbicidovorans MH proved to be identical. However, when RdpA [(R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate dioxygenase] was investigated after purification from the various strains, significant differences in the kinetics and some chemical properties of the enzymes were observed. The preference for substrates ranged in the order (R)‐2‐(2,4‐dichlorophenoxy)propionate (2,4‐DP) > (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate (MCPP) >> 2,4‐dichlorophenoxyacetate (2,4‐D) ∼ 4‐chloro‐2‐methylphenoxyacetate (MCPA), but detailed kinetic investigations revealed significant strain‐dependent differences in the kcat and KM values. While the KM values of RdpA from the various strains were low and their range rather narrow with 2,4‐DP (19–60 μM) and MCPP (35–64 μM), larger differences were observed with phenoxyacetates which were distinctly higher and spanned a wider range with 2,4‐D (237–935 μM) and MCPA (164–510 μM). The lowest KM values with 2,4‐D and MCPA were found for RdpA originating from strain P230. Investigation of the enzymes from the various sources by 2D gel electrophoresis revealed up to three monomeric enzyme forms which differed in the pI value. The 2D‐patterns were similar with RdpA from strains MC1 and MH, and after heterologous expression of the enzyme in Escherichia coli, but differed significantly from that of strain P230. The presence of enzyme forms and their different composition coincided apparently with the differences observed in the kinetic properties of RdpA in the various strains. The effects are discussed in terms of posttranslational modification of RdpA which appears to be different in extent and kind in the various strains.
Two alpha-ketoglutarate-dependent dioxygenases carrying enantiospecific activity for the etherolytic cleavage of racemic phenoxypropionate herbicides [(RS)-2-(2,4-dichlorophenoxy)propionate and (RS)-2-(4-chloro-2-methylphenoxy)propionate] from Delftia acidovorans MC1 were characterized with respect to protein and sequence data. The (S)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (SdpA) appeared as a monomeric enzyme with a molecular weight of 32 kDa in the presence of SDS. N-terminal sequences revealed relationship to alpha-ketoglutarate-dependent taurine dioxygenase (TauD) and to 2,4-dichlorophenoxyacetate/alpha-ketoglutarate-dioxygenase (TfdA). The (R)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (RdpA) referred to 36 kDa in the presence of SDS and to 108 kDa under native conditions. Internal sequences of fragments obtained after digestion made evident relationship to TfdA and TauD. Two-dimensional electrophoretic separation resulted in the resolution of up to 3 individual spots with almost identical molecular weights but different isoelectric points with both RdpA and SdpA. The structural differences of these isoenzyme forms are not yet clear.
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