Involvement of two alpha-ketoglutarate-dependent dioxygenases in enantioselective degradation of (R)- and (S)-mecoprop by Sphingomonas herbicidovorans MH

Abstract

Cell extracts of Sphingomonas herbicidovorans MH grown on (R)-mecoprop contained an enzyme activity that selectively converted (R)-mecoprop to 4-chloro-2-methylphenol, whereas extracts of cells grown on (S)-meco-prop contained an enzyme activity selective for the S enantiomer. Both reactions were dependent on ␣-ketoglutarate and ferrous ions. Besides 4-chloro-2-methylphenol, pyruvate and succinate were detected as products of the reactions. Labeling experiments with 18 O 2 revealed that both enzyme activities catalyzed a dioxygenation reaction. One of the oxygen atoms of pyruvate and one of the oxygen atoms of succinate were derived from molecular oxygen. Analysis of cell extracts obtained from cells grown on different substrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that growth on (R)-mecoprop and (S)-mecoprop caused the appearance of prominent protein bands at 34 and 32 kDa, respectively. Both protein bands were present when cells grew on the racemic mixture. The results demonstrate that S. herbicidovorans initiated the degradation of each enantiomer of mecoprop by a specific ␣-ketoglutarate-dependent dioxygenase. By comparing conversion rates of various phenoxy herbicides, we confirmed that the two enzyme activities were distinct from that of TfdA, which catalyzes the first step in the degradation of 2,4-dichlorophenoxyacetic acid in Ralstonia eutropha JMP134.Substituted phenoxyalkanoic acid herbicides are widely used in weed control. 2,4-Dichlorophenoxyacetic acid (2,4-D) and the chiral compounds (R,S)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) and (R,S)-2-(2,4-dichlorophenoxy)propionic acid (dichlorprop) are members of this class of herbicides. The herbicidal activities of the last two compounds are exclusively associated with the R enantiomers. Today, the pure R enantiomers of mecoprop and of dichlorprop are commercially available as mecoprop-P and dichlorprop-P, respectively. Yet many commercial formulations contain the racemic mixtures and not the pure R enantiomers (27). In order to understand the environmental fate of the chiral compound mecoprop, it is important to study the degradation of the enantiomers independently, as has been recently emphasized (1,2,9,22,29).Studies concerning the fate and the persistence of phenoxyalkanoic acids in soil revealed that degradation of these herbicides is caused mainly by microorganisms (17, 23). The breakdown of 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2,4-D starts with the removal of the alkanoic acid side chain to yield the corresponding phenol (17). Consequently, a great deal of research effort was put into unravelling the subsequent degradation of 2,4-dichlorophenol and 4-chloro-2-methylphenol, the metabolites of 2,4-D and MCPA, respectively (10). In soil, the microbial metabolism of mecoprop leads to the appearance of 4-chloro-2-methylphenol (24). This metabolite is also formed in a mixed bacterial culture with mecoprop as the sole source of carbon and energy (14). So far, only two pure bacterial strains have be...

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

“…Besides the enzymes of strain MC1, there are strong indications of enantioselective enzymes with respect to 2-phenoxypropionates in the case of S. herbicidovorans MH. The separation of crude extracts of this strain by SDS electrophoresis revealed distinct protein bands of 32 kD and 34 kD after growth on (S)-MCPP or (R)-MCPP, respectively, and of both of these bands after growth on the racemate [14]. This pattern tallies with the molecular weights found for the respective ether-cleaving proteins (subunits) of strain MC1.…”

“…Again, this reaction proved to be catalysed by an α-ketoglutarate-dependent dioxygenase [14][15][16]. Preliminary investigations revealed enantioselective properties of the enzymes catalysing the cleavage of the ether bond of the racemic phenoxypropionates as holds for S. herbicidovorans MH [14,17] and D. acidovorans MC1 [15]. The enantioselectivity of this reaction is also supported by the fact that a strain of Alcaligenes denitrificans is only able to utilise the R-enantiomer of 2-(4-chloro-2-methylphenoxy)propionate [18].…”

“…Remarkably, these strains utilise a broader spectrum of phenoxy herbicides; in addition to their ability of degrading the racemic phenoxypropionates, they similarly attack phenoxyacetate derivatives. Again, this reaction proved to be catalysed by an α-ketoglutarate-dependent dioxygenase [14][15][16]. Preliminary investigations revealed enantioselective properties of the enzymes catalysing the cleavage of the ether bond of the racemic phenoxypropionates as holds for S. herbicidovorans MH [14,17] and D. acidovorans MC1 [15].…”

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

“…[4][5][6][7][8][9][10][11][12] Strains selected for their capability to utilize racemic phenoxypropionate herbicides like (RS)-2-(2,4-dichlorophenoxy)propionate (2,4-DP, dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy)propionate (MCPP, mecoprop) are more rare. Examples are Sphingobium herbicidovorans MH, 8,[13][14][15] Delftia acidovorans MC1, 16,17) Rhodoferax sp. P230, 17,18) and several less well described strains.…”

The recalcitrance of clofibric acid to microbial degradation

“…It is a structural isomer of clofibric acid, differing only in the position of one methyl group. Various types of microorganisms including Sphingomonas herbicidovorans are known to be able to degrade (R)-mecoprop via cleavage of the ether bond [7]. Metabolites in the biodegradation process include 4-chloro-2-methylphenol and pyruvate [7].…”