A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression

Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.     

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: purification and characterization

Dicamba O-demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase(DIC) is a homotrimer (alpha)3 with a subunit molecular mass of approximately 40 kDa. FerredoxinDIC and reductaseDIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe-2S](2+/1+) cluster in ferredoxinDIC and a single Rieske [2Fe-2S](2+; 1+) cluster within oxygenaseDIC. Consistent with the presence of a Rieske iron-sulfur cluster, oxygenaseDIC displayed a high reduction potential of E(m,7.0) = -21 mV whereas ferredoxinDIC exhibited a reduction potential of approximately E(m,7.0) = -171 mV. Optimal oxygenaseDIC activity in vitro depended on the addition of Fe2+. The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O-demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenaseDIC is an important new member of the Rieske non-heme iron family of oxygenases.     

Specificity of aFlavobacterium in the metabolism of substituted chlorobenzoates

Summary A strain ofFlavobacterium breve capable of utilizing 3,5-dichlorosalicylate as a sole source of carbon and energy was identified. Degradation of 3,5-dichlorosolicylate, was specific as this strain did not metabolize dicamba (3,6-dichloro-2-methoxybenzoic acid), 3,5-dicamba (3,5-dichloros2-methoxybenzoic acid), or 3,6-dichlorosalicylate. The organism was able to remove completely 3,5-dichlorosalicylate in the presence of three times as much 3,6-dichlorosalicylate being degraded. The organism was able to utilize 3,5-dichlorosalicylate at concentrations up to 1000g/ml. A mixture of 3,5 and 3,6-dichlorosalicylate isomers purified by biological destruction of the unwanted isomer (3,5-dichlorosalicylate) would be useful for producing isomerically pure dicamba, an important herbicide.     

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli

The effects of the herbicides 1,1'-dimethyl-4,4'-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1muM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.     

Effect of herbicide banvel on rabbit vaginal mucus membrane

Effect of banvel and its active ingredient, dicamba (3,6-dichloro-o-anisic acid) was investigated employing rabbit mucus membrane irritancy test. Inflammatory changes which did not exceed an average score of 2+ were observed in the animals 48 hr after a single intravaginal application of banvel (0.1 ml/rabbit) and dicamba (100 mg/rabbit). Persistent histopathological changes were observed in 1 out of 6 banvel-treated rabbits 15 days post-application. The results suggest that banvel and dicamba are not primary irritants but should nevertheless be employed with caution.     

Plasmid-mediated catabolism of dicamba by Pseudomonas species strain PXM

Pseudomonas species strain PXM, which is able to use dicamba (3,6-dichloro-2-methoxybenzoic acid; CAS 1918-00-9, Banvel) as its sole carbon source for growth, has been isolated. The catabolism of dicamba and some of its putative metabolic descendants correlates with the presence of a large and unstable plasmid.     

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics

The herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate putative mechanisms of action and to compare its toxicity with 2-chlorobenzoic acid. Dicamba (4 micro mol/mg mitochondrial protein) induces a limited stimulation of state 4 respiration of ca. 10%, and the above concentrations significantly inhibit respiration, whereas 2-chlorobenzoic acid maximally stimulates state 4 respiration (ca. 50%) at about 25 micro mol/mg mitochondrial protein. As opposed to these limited effects on state 4 respiration, transmembrane electrical potential is strongly decreased by dicamba and 2-chlorobenzoic acid. Dicamba (25 micro mol/mg mitochondrial protein) collapses, almost completely, Deltapsi; similar concentrations of 2-chlorobenzoic acid promote Deltapsi drops of about 50%. Proton permeabilization partially contributes to Deltapsi collapse since swelling in K-acetate medium is stimulated, with dicamba promoting a stronger stimulation. The Deltapsi decrease induced by dicamba is not exclusively the result of a stimulation on the proton leak through the mitochondrial inner membrane, since there was no correspondence between the Deltapsi decrease and the change on the O(2) consumption on state 4 respiration; on the contrary, for concentrations above 8 micro mol/mg mitochondrial protein a strong inhibition was observed. Both compounds inhibit the activity of respiratory complexes II and III but complex IV is not significantly affected. Complex I seems to be sensitive to these xenobiotics. In conclusion, dicamba is a stronger mitochondrial respiratory chain inhibitor and uncoupler as compared to 2-chlorobenzoic acid. Apparently, the differences in the lipophilicity are related to the different activities on mitochondrial bioenergetics.     

Translocation of the Herbicide Dicamba in Purple Nutsedge, Cyperus rotundus

Pairs of nutsedge plants connected by rhizomes were planted, each in a separate pan, without cutting the rhizomes. One plant within each pair was treated with 5.7 kg of dicamba per hectare seven days later. Ten days after treatment, the treated plants and those plants attached to them by rhizomes were harvested separately and analyzed by gas chromatography. The attached plants were found to contain 6 per cent as much dicamba as did the directly treated plants. The suspected metabolites, 3,6-dicblorosalicylic acid, 3,6-dicblorogentisic acid and 5-HO-dicamba, were not detected.     

Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 Å resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.     

Participation of ethylene in common purslane response to dicamba

The responses of common purslane (Portulaca oleracea L.) plants to 2-methoxy-3,6-dichlorobenzoic acid (dicamba) were found to be similar in many respects to ethylene fumigation effects. Dicamba and ethylene increased the permeability of cell membranes in purslane tissues. An increased efflux of electrolytes was observed in the bending region of the stems of dicamba-treated plants. Epinastic leaves after dicamba (10 micrograms) and ethylene (microliter per liter) treatments showed an increased efflux of rubidium. The permeability effects were observable within 1 day after dicamba or ethylene application. Protein metabolism in purslane leaves was not influenced by dicamba until 2 days after treatment, as indicated by reduced nitrate reductase activity. Inhibition of phenylalanine-U-¹⁴C incorporation into protein was observed 3 days after treatment. Ethylene reduced both phenylalanine-U-¹⁴C incorporation into protein and nitrate reductase activity within 1 day. Dicamba caused a rapid increase in ethylene production in purslane plants to levels many times greater than those observed in untreated plants. It was concluded that the dicamba-enhanced production of ethylene is responsible for many of the observed effects of the herbicide.     

Engineering dicamba selectivity in crops: a search for appropriate degradative enzyme(s)

The biotechnologial approaches to conferring crop selectivity to herbicides have been demonstrated for a number of compounds such as glyphosate, glufosinate, imidazolinones and cyclohexanediones. Imidazolinone-resistant and cyclohexanedione-resistant maize lines are already in the market. There are several other effective and environmentally benign herbicides such as dicamba, for which engineering crop selectivity is desirable, to broaden the product utility in different crops and provide new solutions for weed control. One of the most effective approaches to conferring dicamba selectivity in crops is to incorporate a gene for its rapid metabolism. It is advantageous to have different dicamba-metabolizing enzymes in order to maximize the chances of at least one functioning optimally in a plant environment. Three different metabolizing enzymes are currently available to engineer crop selectivity. The first one is the folate-dependent O-demethylase from Clostridium thermoaceticum, that converts dicamba to herbicidally inactive 3,6-dichlorosalicylate. The second enzyme is the NADH-dependent, multi-component monooxygenase from Pseudomonas maltophilia DI-6 that also converts dicamba to 3,6-dichlorosalicylate. The third enzyme is from corn endosperm cultures that catalyzes the 5-hydroxylation of dicamba. The merits of these three enzymes are discussed with respect to conferring crop selectivity to dicamba. In addition, a rapid microbial screen was conceived for discovery of new dicamba-degrading bacteria, which resulted in identification of Pseudomonas orvilla. This bacteria degraded dicamba by the same pathway, perhaps using a similar enzyme system as Pseudomonas maltophilia DI-6. However, the microbial screen has the potential to identify novel bacteria that degrade dicamba by a different pathway, providing more options for metabolizing enzymes to confer herbicide selectivity in crops. Received 13 February 1997/ Accepted in revised form 26 June 1997     

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.     

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O₂ into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (α₃) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co²⁺, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 Å, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.     

A small-scale,inexpensive method for detecting formaldehyde or methanol in biochemical reactions containing interfering substances

A simple, inexpensive microdistillation device is described for capturing methanol or formaldehyde as end products of biochemical reactions or in environmental samples. We demonstrate that the microdistillation protocol, coupled with the use of alcohol oxidase and the formaldehyde-sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), serves as a quick and inexpensive alternative to chromatographic and mass spectrometer analyses for determining if formaldehyde or methanol is a product of reactions that contain substances that interfere with the Purpald reaction. These techniques were used to affirm formaldehyde as the end product of the dicamba monooxygenase-catalyzed O-demethylation of the herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid).     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Application of the disk method: responses to growth regulators

The paper disk method of screening several plant growth regulators was evaluated. Leaf explants ofVigna unguiculata (L) Walp. were placed on solidified Murashige and Skoog's minimal organics medium containing 0.5 mg/l nicotinic acid. Hormones were tested, singly and in combinations, on paper disks in large Petri plates (150×20 mm). Hormones tested were 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), picloram (4-amino-3,5,6-trichloropicolinic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid), BA (6-benzyladenine), 2iP (2-isopentenyl adenine), and kinetin [6-(furfurylamino)-purine]. Root formation was stimulated by IAA and IBA; dicamba, picloram, 2,4-D, and 2,4,5-T stimulated callus formation. All cytokinins tested suppressed root formation. Dicamba in combination with either 2iP or kinetin induced the greatest callus formation. Root formation was optimal with kinetin and either IAA or IBA. The disk method provided a rapid, nonquantitative evaluation of callus and root formation from leaf disks.     

Comparative Effects and Concentration of Picloram, 2,4,5-T and Dicamba in Tissue Culture

In nutrient agar comparative concentrations (10^?3 to 10^?5M) of (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) were generally more inhibitory to the growth of tissue cultures of soybean (Glycine max (L.) Merrill cv. Acme) and cottonwood (Populus deltoides Marsh.) than were either 4-amino-3,5,6-trichloropicolinic acid (picloram) or 3,6-dichloro-o-anisic acid (dicamba). Compared to untreated tissue dicamba or picloram at 10^?6M in the nutrient agar resulted in a 200 % increase in the growth of soybean tissue. At 10^?5 and 10^?6M dicamba also produced an increase in the growth of cottonwood tissue. Greatest absorption of picloram and dicamba by tissue cultures from agar occurred during the first 24 h after treatment. However, absorption remained nearly static thereafter for 14 days. More dicamba was absorbed by soybean and cottonwood tissue cultures than either picloram or 2,4,5-T.     

Effect of nitrogen and phosphorus status on the translocation of three herbicides in field bindweed (Convolvulus arvensis L.)

Twenty-eight day old field bindweed plants grown in culture solutions deficient in nitrogen (N) or phosphorus (P) for the last seven days of growth translocated significantly less foliarly applied dicamba (3,6-dichloro-o-anisic acid) and 2,4-D [(2,4-dichlorophenoxy) acetic acid] to their roots than did plants grown in complete nutrient solutions. In contrast, N deficiency stimulated basipetal translocation of glyphosate [N-(phosphonomethyl) glycine] and inhibited its acropetal translocation in field bindweed. Deficiencies of both N and P decreased translocation of dicamba from the treated area, but had no influence on translocation of glyphosate or 2,4-D from the treated area.Journal Article No. 4406 of the Agric. Exp. Stn., Oklahoma State University.     

Modeling Dicamba Sorption and Transport Through Zoysiagrass Thatch and Soil

The presence of turfgrass thatch complicates the sorption and transport of water soluble pesticides because the surface-applied pesticides must pass through an organic-rich thatch layer prior to entering the soil. The study was conducted (1) to determine the impact of zoysiagrass thatch (Zoy-sia japonica Steud.) on dicamba (3,6-dichloro-2-methoxy benzoic acid) transport through soil columns, and (2) to evaluate the effectiveness of linear equilibrium (LEM), two site nonequilibrium (2SNE) and one site nonequilibrium (1SNE) models to predict dicamba transport through columns containing a surface layer of thatch and columns devoid of thatch. The equilibrium sorption isotherms of ¹⁴C dicamba to homogenized samples of zoysiagrass thatch and a Sassafras loamy sand soil (fine loamy, mixed mesic, Typic Hapludult) were determined. Following the application of bromide to determine transport parameters, 0.56?kg dicamba ha^?1 was surface applied to undisturbed soil columns containing a surface layer of thatch and columns devoid of thatch and leachate samples collected for 12?h under steady-state unsaturated conditions. Zoysiagrass thatch (Kf = 0.82) had a three times greater sorption capacity than the soil (Kf = 0.28) beneath the thatch. Dicamba leaching for columns with thatch layers was ca. 21% less than soil columns devoid of thatch. When dicamba breakthrough curves were fitted to the different forms of the convective dispersive equation, the 2SNE model simulated dicamba transport better than LEM and 1SNE models, indicating the presence of two-site nonequilibrium sorption. Indications are that turfgrass thatch may have significant effects on dicamba leaching that presently used regulatory models based on LEM approach do not adequately consider.     

Degradation of dicamba by an anaerobic consortium enriched from wetland soil.

The biodegradability of dicamba was investigated under anaerobic conditions with a consortium enriched from wetland soil. Degradation proceeded through an initial demethylation reaction, forming 3,6-dichlorosalicylic acid, followed by reductive dechlorination, forming 6-chlorosalicylic acid. The consortium, consisting of a sulfate reducer, three methanogens, and a fermenter, was unable to mineralize the aromatic ring.