The Catalytic Mechanism Of Dicamba Methyltransferase Dmt50 And Its Potential Application In Herbicide Resistant Transgenic Engineering

The application of genetically modified(GM)herbicide-resistant crops significantly increased the efficiency of weed control and resulted in great economic benefits.Currently,glyphosate-resistant transgenic crops are the most widely used.However,long-term use of glyphosate has resulted in severe weed resistance.Therefore,it is urgent to find new target herbicides and develop corresponding GM herbicide-resistant crops to combat glyphosateresistant weeds.Dicamba is a benzoic acid herbicide.Because of its advantages of high efficiency,low toxicity,low cost and slow development of weed resistance,dicamba is considered as an ideal target herbicide for constructing transgenic crops.The dicambaresistant GM crops developed by Monsanto using the dicamba monooxygenase gene dmo has been has been commercialized,with an annual promotion area of more than 200 million mu.In contrast,the current research on herbicide-resistant transgenes is still weak for China,and the lack of herbicide-resistant gene(enzyme)resources with independent intellectual property rights has seriously affected the development and application of herbicide resistant transgenic breeding.In our previous study,a dicamba degradation strain,Rhizorhabdus dicambivorans Ndbn-20,was isolated and a dicamba methyltransferase gene dmt50,which has potential transgenic application value was cloned from this strain.Unlike the monooxygenase DMO,Dmt50 is a THF(tetrahydrofolate)dependent methyltransferase.In this study,In this study,the enzymatic properties,structure and catalytic mechanism of Dmt50 were studied,and mutant with increased enzyme activity were obtained through directed evolution screening,and the dicamba resistance of dmt50 transgenic Arabidopsis thaliana was evaluated.The main research results are as follows:1.The enzymatic properties of dicamba methyltransferase dmt50Dmt50 was expressed in Escherichia coli BL21(DE3)using pET29a(+)expression vector and purified by affinity chromatography to obtain pure enzyme for further study.enzyme assays showed that,in the presence of THF(tetrahydrofolate),Dmt50 can transfer the methyl group of dicamba to the N5 of THF methotrexate ring to produce 5-methyl-THF(5-methyl-tetrahydrofolate)and 3,6-DCSA(3,6-dichlorosalicylic acid).This reaction was feedback inhibited by product 5-methyl-THF,and the final conversion rate of dicamba was about 35%.Using Mthfr(methylenetetrahydrofolate reductase)to further convert 5-methylTHF can release 5-methyl-THF feedback inhibition on Dmt50 and dicamba can be completely converted.Under the condition that the product inhibition is released,in a 300 μL reaction system,the kinetic parameters of dicamba methyl transfer reaction catalyzed by 0.1 mg Dmt50 are Vmax=0.041 mM/min,Km=0.247 mM,kcat=6.589 min-1 and kcat/Km=26.698 min-1·mM-1,respectively.2.Crystal structure and catalytic mechanism of dicamba methyltransferase Dmt50Through the screening and optimization of protein crystallization conditions,the Dmt50 protein crystals and Dmt50-5-methyl-THF crystals were obtained,and the structure of Dmt50 has been resolved by homologous modeling.Then,Dmt50-5-methyl-THF complex crystals was also obtained based on the original crystallization conditions of Dmt50.By comparing these two structures,we found that Ile28-Thr39 region at the N-terminal of Dmt50 is a flexible area.After Dmt50 is combined with 5-methyl-THF,this flexible region forms an αhelix and closes one side of the central cavity to form a catalytic pocket.The results of dicamba-Dmt50 molecular docking show that this region is involved in the positioning of the substrate dicamba and may affect the way the substrate enters.In the process of Dmt50 catalyzing the methyl transfer of dicamba,dicamba first enters the active center,and then THF enters;after THF binds to Dmt50,it triggers a conformational change in the flexible region of Dmt50 to form a-helix,which locks dicamba in the active center;Tyr253 and His69 forms a hydrogen bond with the oxygen atom in the methoxy group of dicamba to protonate the oxygen atom and activate the methyl group;the lone pair of electrons on N5 of THF carries out a nucleophilic attack on the activated methyl group and completes the methyl transfer to form 3,6-DCSA and 5-methyl-THF.3.Screening high enzyme activity Dmt50 mutantBase on the chromogenic reaction of 3,6-DCSA,a set of screening methods for high enzyme activity Dmt50 mutants were established.Finally,a Dmt50 mutant(Mutant)with a 15.6%increase in enzyme activity was obtained by site-directed saturation mutation and directed evolution screening.This mutant contains a large number of mutation sites,but only 4 mutation sites(F32Y,I43V,M263I,I318L)are located in key regions(Ile28-Thr39 flexible region,active center region,and folate binding region).In order to study the effects of these site mutations on enzyme activity,Dmt50 F32Y-I43V-M263I-I318L mutant and Mutant F32Y-I43V-M263I-I318L reverse mutant were further constructed.The results of enzyme assays showed that these 4 site mutations are necessary to increase the enzyme activity of the Mutant,but only these 4 site mutations did not help to increase the enzyme activity of Dmt50.The mechanism of the increased enzyme activity of the Dmt50 mutant may be due to the combined effect of a large number of site mutations,which reduces the affinity of 5-methylTHF and partially relieves the product inhibition and ultimately increases the conversion rate of dicamba.4.A.thaliana carrying dmt50 gene exhibited resistance to dicambaThe dmt50 transgenic A.thaliana was constructed using the dmt50 gene,and its dicamba resistance was evaluated.The results of resistance assay in plate showed that in the treatment of 1 mg/L dicamba,the root development of transgenic A.thaliana was normal,the leaves only showed slight curl,and the fresh weight was significantly higher than that of wild type A.thaliana(P<0.05),The results of resistance assay in soil(dicamba spraying treatment)showed that the dmt50 transgenic A.thaliana can tolerate the dicamba spraying treatment at 560 g/ha(normal field application concentration),but died under 1120 g/ha.In order to further improve the dicamba resistance of transgenic A.thaliana,the mthfr gene and dmt50 gene were co-expressed to construct dmt50+mthfr66 cotransgenic A.thaliana:The results of resistance assay in plate showed that,under the treatment of 1 mg/L dicamba,the growth of co-transgenic A.thaliana has been basically the same as that of treatment without dicamba(positive control).The results of resistance assay in soil(dicamba spraying treatment)showed that under 560g/ha(normal field application rate)dicamba treatment,the root length and fresh weight of cotransgenic A.thaliana were significantly higher than those of dmt50 transgenic A.thaliana(P<0.05)and cotransgenic A.thaliana can tolerate 1120 g/ha(2xnormal field application rate)dicamba treatment.In summary,the development of this study is conducive to deepening the understanding of the catalytic mechanism of THF dependent demethylase;it also provides technical support for the application of dicamba demethylase with independent intellectual property rights.