The two enantiospecific dichlorprop/α-ketoglutarate-dioxygenases from Delftia acidovorans MC1 – protein and sequence data of RdpA and SdpA

Abstract

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.

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

“…Irreversible metabolic steps are particularly decisive to channel the compound into general metabolism. [52][53][54] With strain MC1, the -ketoglutarate-dependent dioxygenases, viz., RdpA and above all SdpA, which initiate the degradation of 2,4-D, should play such a role (for details of the enzyme data, see Westendorf et al 23,24) ). The cleavage reaction depends on the presence ofketoglutarate, the supply of which is strongly coupled to the general metabolism and to the physiological state of the population.…”

“…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.…”

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

“…Proteolytic modifications should be ruled out as the cause of this pattern as this was also observed in enzyme preparations carried out in the presence of protease inhibitors. However, two-dimensional electrophoretic separation of the respective enzyme preparations or applying crude extracts revealed protein spots which are indicative of isoforms differing in charge rather than molecular weight [25]. This has to be verified in order to elucidate the structure and catalytic properties of these isoenzymes.…”

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).…”