Dicamba Monooxygenase Research Articles

Risk assessment of homologous variants of biotech trait proteins using a bridging approach

ABSTRACT A transgenic protein is frequently expressed as different homologous variants in genetically modified crops due to differential processing of targeting peptides or optimization of activity and specificity. The aim of this study was to develop a science-based approach for risk assessment of homologous protein variants using dicamba mono-oxygenase (DMO) as a case study. In this study, DMO expressed in the next-generation dicamba-tolerant maize, sugar beet and soybean crops exhibited up to 27 amino acid sequence differences in the N-terminus. Structure modeling using AlphaFold, ESMFold and OpenFold demonstrates that these small N-terminal extensions lack an ordered secondary structure and do not disrupt the DMO functional structure. Three DMO variants were demonstrated to have equivalent immunoreactivity and functional activity ranging from 214 to 331 nmol/min/mg. Repeated toxicity studies using each DMO variant found no test substance-related adverse effects. These results support that homologous protein variants, which have demonstrated physicochemical and functional equivalence, can leverage existing safety data from one variant without requiring additional de novo safety assessments.

Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM.

Rieske nonheme iron oxygenases use two metallocenters, a Rieske-type [2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation reactions on a broad range of substrates. These enzymes are widely used by microorganisms to degrade environmental pollutants and to build complexity in a myriad of biosynthetic pathways that are industrially interesting. However, despite the value of this chemistry, there is a dearth of understanding regarding the structure-function relationships in this enzyme class, which limits our ability to rationally redesign, optimize, and ultimately exploit the chemistry of these enzymes. Therefore, in this work, by leveraging a combination of available structural information and state-of-the-art protein modeling tools, we show that three "hotspot" regions can be targeted to alter the site selectivity, substrate preference, and substrate scope of the Rieske oxygenase p-toluenesulfonate methyl monooxygenase (TsaM). Through mutation of six to 10 residues distributed between three protein regions, TsaM was engineered to behave as either vanillate monooxygenase (VanA) or dicamba monooxygenase (DdmC). This engineering feat means that TsaM was rationally engineered to catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than its favored native para position, and that TsaM was redesigned to perform chemistry on dicamba, a substrate that is not natively accepted by the enzyme. This work thus contributes to unlocking our understanding of structure-function relationships in the Rieske oxygenase enzyme class and expands foundational principles for future engineering of these metalloenzymes.

Assessment of genetically modified maize MON 87419 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2017-140).

Genetically modified maize MON 87419 was developed to confer tolerance to dicamba- and glufosinate-based herbicides. These properties were achieved by introducing the dmo and pat expression cassettes. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between maize MON 87419 and its conventional counterpart needed further assessment, except for the levels of arginine and protein in grains which did not raise safety and nutritional concerns. The GMO Paneldoes not identify safety concerns regarding the toxicity and allergenicity of the dicamba mono-oxygenase (DMO) and phosphinothricin N-acetyltransferase (PAT) proteins as expressed in maize MON 87419. The GMO Panelfinds no evidence that the genetic modification impacts the overall safety of maize MON 87419. In the context of this application, the consumption of food and feed from maize MON 87419 does not represent a nutritional concern in humans and animals. The GMO Panelconcludes that maize MON 87419 is as safe as the conventional counterpart and non-GM maize varieties tested, and no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable maize MON 87419 grains into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 87419. The GMO Panelconcludes that maize MON 87419 is as safe as its conventional counterpart and the tested non-GM maize varieties with respect to potential effects on human and animal health and the environment.

Assessment of genetically modified oilseed rape MON 94100 for food and feed uses, under regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2020-169).

Oilseed rape MON 94100 was developed to confer tolerance to dicamba herbicide. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between oilseed rape MON 94100 and its conventional counterpart needs further assessment, except for the levels of carbohydrates, calcium and ADF in seeds, which do not raise nutritional and safety concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the dicamba mono‐oxygenase (DMO) protein as expressed in oilseed rape MON 94100. The GMO Panel finds no evidence that the genetic modification impacts the overall safety of oilseed rape MON 94100. In the context of this application, the consumption of food and feed from oilseed rape MON 94100 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that oilseed rape MON 94100 is as safe as the conventional counterpart and non‐GM oilseed rape reference varieties tested, and no post‐market monitoring of food/feed is considered necessary. In the case of accidental release of viable oilseed rape MON 94100 seeds into the environment, this would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of oilseed rape MON 94100. The GMO Panel concludes that oilseed rape MON 94100 is as safe as its conventional counterpart and the tested non‐GM oilseed rape reference varieties with respect to potential effects on human and animal health and the environment.

AGROBACTERIUM-MEDIATED TRANSFORMATION OF COTTON (GOSSYPIUM HIRSUTUM L.) USING DMO GENE FOR ENHANCED TOLERANCE AGAINST DICAMBA PESTICIDE

The agrobacterium based transformation of herbicide-resistant crops has modernized weed management in crops by producing cost-effective and ecosystem friendly transgenic plants. Cotton is one of the major crops which are grown worldwide due to its great economic value in textile industries. Dicamba is a commonly used herbicide in broadleaf plants to kill a wide range of weeds in many dicotyledonous crop fields since the 20th century. In this study, Eagle 2 cotton variety was transformed with the DMO gene which is responsible for the synthesis of the Dicamba monooxygenase enzyme that exhibits tolerance against the Dicamba herbicide. This entire study was conducted at Four Brothers Genetics Lab, Lahore. Transformed cultures of Agrobacterium Tumefaciens with the DMO gene were acquired. Cotton embryos were isolated and co-cultivated with transformed Agrobacterium cultures under sterile conditions. Transformed embryos were grown on an artificial growth medium and acclimatized under favorable conditions. Healthy and stable plants were shifted infield where they were grown into a mature plant. Leaf samples of these plants were collected and DNA was successfully isolated by the CTAB method. Transformed plants were confirmed by Polymerase chain reaction and gel electrophoresis. Variations in different traits among transformed cotton plants were found which indicated that the transgenic plant 4 showed higher plant height, monopodial and sympodial branches, leaf length, leaf width, number of bolls, and bolls weight. The better performance of plant 4 indicated that the yield potential of the transgenic plant was improved as compared with other transgenic cotton plants.

Effect of common processing of soybeans on the enzymatic activity and detectability of the protein, Dicamba Mono-Oxygenase (DMO), introduced into dicamba-tolerant MON 87708

Assessing the safety of genetically engineered crops includes evaluating the risk (hazard and exposure) of consuming their newly expressed proteins. The dicamba monooxygenase (DMO) protein, introduced into soybeans to confer tolerance (DT) to dicamba herbicide, was previously characterized and identified to pose no food or feed safety hazards. Most agricultural commodities (e.g., soybeans, maize) enter the food supply after processing methods that can include exposure to high temperatures, harsh solvents or pH extremes that can adversely impact the structure and function of proteins. To understand the likelihood of exposure to DMO in foods from DT soy, enzymatically active and/or immunodetectable forms of DMO were measured in pilot-scale productions of two soy foods (soymilk and tofu), and eight processed fractions (full fat flour, inactivated full fat flour, defatted flour, toasted meal, protein isolate, protein concentrate, crude lecithin, and refined, bleached and deodorized oil). Western blot analysis detected DMO in tofu and in five of the eight processed fractions. DMO activity was not detected in either soymilk or tofu, nor in six of the eight processed fractions. Therefore, many commercial soy processing methods can denature and/or degrade introduced proteins, like DMO. Although the DMO protein has shown no evidence of hazard, this study demonstrates that processing further reduces any food or feed risk by limiting dietary exposure to intact DMO protein.

Scientific opinion on an application by Monsanto (EFSA-GMO-NL-2013-114) for the placing on the market of a herbicide-tolerant genetically modified cotton MON88701 for food and feed uses, import and processing under Regulation (EC) No1829/2003.

Cotton MON 88701 was developed through Agrobacterium tumefaciens‐mediated transformation to express the dicamba mono‐oxygenase (DMO) protein, conferring tolerance to dicamba, and the phosphinothricin N‐acetyltransferase PAT protein, conferring tolerance to glufosinate ammonium‐based herbicides. The molecular characterisation data and bioinformatics analyses did not identify issues requiring further assessment for food/feed safety. The agronomic and phenotypic characteristics tested revealed no relevant differences between cotton MON 88701 and its conventional counterpart. Since complete compositional results were reported for only three sites, the EFSA Panel on Genetically Modified Organisms (GMO Panel) is not in the position to complete the assessment of the compositional analysis. Moreover, as no 28‐day toxicity study in rodents on the MON 88701 DMO protein was provided, the GMO Panel is not in the position to complete the safety assessment of this protein in cotton MON 88701. Consequently, the GMO Panel cannot complete the toxicological, allergenicity and nutritional assessment of food/feed derived from cotton MON 88701. The safety assessment identified no concerns regarding the potential toxicity and allergenicity of the PAT protein newly expressed in cotton MON 88701. Considering the routes of exposure and limited exposure levels, the GMO Panel concludes that cotton MON 88701 would not give rise to safety concerns in the event of accidental release of viable seeds into the environment. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of cotton MON 88701. In conclusion, in the absence of an appropriate comparative assessment and an appropriate assessment of the MON 88701 DMO protein, the GMO Panel is not in a position to complete its food/feed risk assessment of cotton MON 88701. The GMO Panel concludes that the cotton MON 88701 is unlikely to have any adverse effect on the environment in the context of the scope of the application.

Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6–97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

A subchronic feeding study of dicamba-tolerant soybean with the dmo gene in Sprague–Dawley rats

The dicamba-tolerant soybean MON87708 expresses the dicamba mono-oxygenase (DMO) enzyme that is encoded by the dmo gene. In order to evaluate the safety of this soybean, a 90-day subchronic feeding toxicity study (13 weeks) was conducted on Sprague–Dawley rats. A total of 140 rats were divided into 7 groups (10/sex/group), including a standard commercial diet control group. The genetically modified (GM) soybean MON87708 and the near isogenic non-GM soybean A3525 were respectively processed to unhulled, full-fat, and heat-treated powder, then mixed into the diet at levels of 7.5%, 15%, and 30% (wt/wt) with the main nutrients of the various diets balanced and then fed to 6 groups. The remaining group of rats fed with a commercial rat diet served as blank control. Some isolated parameters indicated statistically significant differences in body weight, feed consumption/utilization, hematology, serum biochemistry, and relative organ weights. These differences were not consistent across gender or test-diet dose, which were attributed to incidental and biological variability. In conclusion, the results demonstrated that the transgenic soybean MON87708 containing DMO was as safe as non-transgenic isogenic counterpart with historical safe use.

Degradation of the herbicide dicamba by two sphingomonads via different O-demethylation mechanisms

Dicamba, a chlorinated benzoic acid herbicide, is widely used to control broadleaf weeds. In this study, two dicamba-degrading sphingomonads Sphingobium sp. Ndbn-10 and Sphingomonas sp. Ndbn-20 were isolated from activated sludge and compost samples, respectively. Both isolates could completely degrade and utilize dicamba, but strain Ndbn-10 possessed relatively higher dicamba degradation efficiency than strain Ndbn-20. The degradation of dicamba in both isolates was initiated by O-demethylation to generate 3,6-dichlorosalicylic acid (DCSA) but through different mechanisms. For Sphingobium sp. Ndbn-10, O-demethylation is catalyzed by a NADH-dependent dicamba monooxygenase (DMO), whereas in Sphingomonas sp. Ndbn-20, a tetrahydrofolic acid (THF)-dependent O-demethylase is responsible for the O-demethylation. Our results suggest that sphingomonads can rapidly and efficiently adapt to degrading dicamba through the evolution of different metabolic pathway mechanisms and highlight a novel THF-dependent dicamba O-demethylation mechanism in bacteria.

Scientific Opinion on application EFSA‐GMO‐NL‐2011‐93 for the placing on the market of the herbicide‐tolerant genetically modified soybean MON 87708 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

Soybean MON 87708 contains a single insert consisting of the dmo expression cassette. The DMO (dicamba mono-oxygenase) proteins confer tolerance to dicamba-based herbicides. Bioinformatic analyses of the inserted DNA and flanking regions do not raise safety issues. The levels of DMO proteins in soybean MON 87708 have been sufficiently analysed. The stability of the genetic modification has been demonstrated. No differences were identified in the compositional data of forage and seeds obtained from soybean MON 87708 or in its agronomic and phenotypic characteristics that would require further assessment with regard to safety by the GMO Panel. The safety assessment identified no concerns regarding the potential toxicity and allergenicity of the DMO proteins, or of soybean MON 87708. The compositional data indicating that soybean MON 87708 is as nutritious as non-GM soybean varieties were supported by the outcome of a chicken study. There are no indications of an increased likelihood of establishment and spread of feral GM soybean plants. Considering the scope of this application, potential interactions of soybean MON 87708 with the biotic and abiotic environment were not considered to be an issue. Environmental risks associated with an unlikely but theoretically possible horizontal gene transfer from soybean MON 87708 to bacteria have not been identified. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean MON 87708. In conclusion, the GMO Panel considers that the information available for soybean MON 87708 addresses the scientific comments raised by the Member States, and that the soybean MON 87708, as described in this application, is as safe as its conventional counterpart and non-GM soybean reference varieties with respect to potential effects on human and animal health or the environment, in the context of its intended uses.

O-Demethylations Catalyzed by Rieske Nonheme Iron Monooxygenases Involve the Difficult Oxidation of a Saturated C–H Bond

Dicamba monooxygenase (DMO) catalyzes the O-demethylation of dicamba (3,6-dichloro-2-methoxybenzoate) to produce 3,6-dichlorosalicylate and formaldehyde. Recent crystallographic studies suggest that DMO catalyzes the challenging oxidation of a saturated C-H bond within the methyl group of dicamba to form a hemiacetal intermediate. Testing of this hypothesis was made possible by our development of two new independent techniques. As a novel method to allow use of (18)O2 to follow reaction products, bisulfite was used to trap newly formed (18)O-formaldehyde in the stable adduct, hydroxymethanesulfonate (HMS(-)), and thereby prevent the rapid exchange of (18)O in formaldehyde with (16)O in water. The second technique utilized unique properties of Pseudomonas putida formaldehyde dehydrogenase that allow rapid conversion of (18)O-formaldehyde into stable and easily detectable (18)O-formic acid. Experiments using these two new techniques provided compelling evidence for DMO-catalyzed oxidation of the methyl group of dicamba, thus validating a mechanism for DMO [and for vanillate monooxygenase, a related Rieske nonheme iron monooxygenase] that involves the difficult oxidation of a saturated C-H bond.

Genetic Engineering of Maize (Zea mays) for High-Level Tolerance to Treatment with the Herbicide Dicamba

Herbicide-tolerant crops have been widely and rapidly adopted by farmers in several countries due to enhanced weed control, lower labor and production costs, increased environmental benefits, and gains in profitability. Soon to be introduced transgenic soybean and cotton varieties tolerant to treatments with the herbicide dicamba offer prospects for excellent broadleaf weed control in these broadleaf crops. Because monocots such as maize (Zea mays) can be treated with dicamba only during a limited window of crop development and because crop injury is sometimes observed when conditions are unfavorable, transgenic maize plants have been produced and tested for higher levels of tolerance to treatment with dicamba. Maize plants expressing the gene encoding dicamba monooxygenase (DMO) linked with an upstream chloroplast transit peptide (CTP) display greatly enhanced tolerance to dicamba applied either pre-emergence or postemergence. Comparisons of DMO coupled to CTPs derived from the Rubisco small subunit from either Arabidopsis thaliana or Z. mays showed that both allowed production of transgenic maize plants tolerant to treatment with levels of dicamba (i.e., 27 kg/ha) greatly exceeding the highest recommended rate of 0.56 kg/ha.

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.

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 O 2 into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (α 3) 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 Co 2+, 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.