Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

Abstract

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).

Uptake Kinetics of 2,4-Dichlorophenoxyacetate byDelftia acidovoransMC1 and Derivative Strains: Complex Characteristics in Response to pH and Growth Substrate

“…This capability is attributed to the expression of two -ketoglutaratedependent dioxygenases, called (R)-2,4-dichlorophenoxypropionate/-ketoglutarate-dioxygenase (RdpA) and (S)-2,4-dichlorophenoxypropionate/-ketoglutarate-dioxygenase (SdpA), which exhibit enantio-and substrate specific properties. 17,23,24) After cleavage of the ether bond, the original substrate becomes anonymous since further metabolism is common in general. This includes hydroxylation of the liberated phenol derivative to the corresponding catechol, e.g., by chlorophenol hydroxylase (TfdB), which is channeled into the general metabolism via enzymes of the modified ortho cleavage pathway, i.e., by TfdC-F. 3,25) Besides ether bond cleavage, transport across the cytoplasmatic membrane has the potential to control herbicide utilization selectively, and above all, if this is facilitated diffusion or active transport.…”

Delftia acidovoransMC1 Resists High Herbicide Concentrations — a Study of Nutristat Growth on (RS)-2-(2,4-Dichlorophenoxy)propionate and 2,4-Dichlorophenoxyacetate

“…20) Reaction was monitored by measuring the initial reaction rate (5 samples within 10 min; almost linear progress curves with R 2 > 0:98) of the liberation of dichlorophenol after the reaction with 4-aminoantipyrine. 21) …”

“…(R)-2,4-DP/ -ketoglutarate-dioxygenase (RdpA) and (S)-2,4-DP/ -ketoglutarate-dioxygenase (SdpA) (see Westendorf et al 21,23) ) were measured to compare their activities to the substrate consumption rates. These rates seemed sufficiently high according to the data in Fig.…”

“…6). It should be mentioned that the activity of RdpA with respect to 2,4-D is low due to high K M and low k cat values 21) and was not considered to contribute to 2,4-D consumption to a significant amount. According to the data in Figs.…”

Resistance of Enlist™(AAD-12) Cotton to Glufosinate

“…Expression of the AAD-12 protein in transgenic crops has been shown to provide tolerance to the herbicide 2,4-D by catalyzing the conversion of 2,4-D to 2,4-dichlorophenol (DCP), a herbicidally inactive compound, through an Fe(II)/α-keto acid-dependent dioxygenase reaction ( Figure 37 and Figure 38) (Müller et al, 1999;Westendorf et al, 2002;Westendorf et al, 2003;Wright et al, 2009;Wright et al, 2010). (Roberts, 1998).…”