Investigation Of Molecular Docking On ESPS Synthase Activity And Resistance To Glyphosate

5-Enolpyruvylshikimate-3-phosphate synthase (EPSP synthase for short), is thesixth enzyme of shikimic acid pathway and participates in the synthesis of aromaticamino acids and some of secondary metabolites. Meanwhile, EPSP synthase is notonly targets of the herbicide (glyphosate), antibiotics, anti-parasitic drugs, but also isimportant regulatory site of promoting the accumulation of shikimic acid in theorganism. In recent years, with the rapid development of molecular biologytechnology and the in-depth study of EPSP synthase, EPSP synthase genes have beenwidely used in resistance to glyphosate for genetically modified crops, medicine andhealth, etc.In this paper, molecular docking on complexes of the Escherichia coli EPSPsynthase·S3P, Agrobacterium tumefaciens sp.CP4EPSP synthase·S3P, Streptococcuspneumoniae EPSP synthase·S3P, Mycobacterium tuberculosis EPSP synthase·S3P,Vibrio cholerae EPSP synthase·S3P and Coxiella burnetii EPSP synthase·S3P withglyphosate and substrate PEP were investigated by using Autodock4.2ofmolecular docking software. The results indicated that the best docking conformationof glyphosate and substrate PEP appeared in the cleft of two structure domains andclosed to S3P; The binding sites of glyphosate and substrate PEP with EPSP synthasewere basically identical and speculated that hydrogenbond, hydrophobic interactionsand electrostatic forces were the main driving force of enzyme binding withglyphosate and substrate PEP; Binding free energies of the six kinds of EPSPsynthase above with PEP were-6.04kcal·mol-1,-6.29kcal·mol-1,-6.10kcal·mol-1,-6.48kcal·mol-1,-6.23kcal·mol-1,-6.11kcal·mol-1, respectively, and the correspondingbinding constants (Km) were37.48?mol,24.36?mol,34.05?mol,17.89?mol,26.93?mol,33.15?mol, respectively, and according to the binding free energies andKm values predicted six kinds of EPSP synthase activity for PEP from high tolow as follows: Mycobacterium tuberculosis, Agrobacterium CP4, Vibriocholerae, Coxiella burnetii, Streptococcus pneumoniae, Escherichia coli; Thebinding free energies of the six kinds of EPSP synthase with glyphosate were-7.30kcal·mol-1,-6.66kcal·mol-1,-6.93kcal·mol-1,-6.73kcal·mol-1,-6.91kcal·mol-1,-6.80k c a l· m o l-1,respectively, and the corresponding inhibition constants (Ki) were4.47?mol,13.22?mol,8.26?mol,11.73?mol,8.60?mol,10.32?mol, respectively, the Km/Ki values were8.38,1.84,4.12,1.53,3.13,3.21, respectively, and according to theKm/Ki values predicted six kinds of EPSP synthase resistance to glyphosate fromstrong to weak as follows: Mycobacterium tuberculosis, Agrobacterium CP4, Vibriocholerae, Coxiella burnetii, Streptococcus pneumoniae, Escherichia coli.Gly residue is a highly conserved amino acid residue of the active site in naturalplant and bacterial EPSP synthase?whereas Ala residue replacing Gly residue is foundat this position in CP4EPSP synthase, so six receptors EPSP synthases based ontheir crystal structure were used to single point mutation with the aid of thePymol software respectively, to get the Gly96Ala mutant EPSP synthase ofEscherichia coli, Ala100Gly mutant EPSP synthase of CP4, Gly92Ala mutant EPSPsynthase of Streptococcus pneumoniae, Gly96Ala mutant EPSP synthase ofMycobacterium tuberculosis, Gly96Ala mutant EPSP synthase of Vibrio cholerae andGly95Ala mutant EPSP synthase of Coxiella burnetii used for molecular docking.Molecular docking on the new receptors of the mutants with glyphosate and substratePEP were investigated. The results indicated that the position of the best dockingconformation of glyphosate, substrate PEP and binding sites were basically identicalbefore and after the mutation, and the main driving force of the mutants EPSPsynthase with glyphosate and substrate PEP binding were still hydrogenbond,hydrophobic interactions and electrostatic forces. The binding free energies of the sixkinds of mutant EPSP synthase above with PEP were-5.85kcal·mol-1,-6.31kcal·mol-1,-5.99kcal·mol-1,-6.36kcal·mol-1,-6.17kcal·mol-1,-6.00kcal·mol-1, respectively, andthe corresponding binding constants (Km) were51.30?mol,23.53?mol,40.94?mol,21.91?mol,29.85?mol,40.31?mol, respectively, and according to the binding freeenergies and Km values predicted six kinds of EPSP synthase mutants activity for PEPfrom high to low as follows: Mycobacterium tuberculosis, Agrobacterium CP4,cholerae, Coxiella burnetii, Streptococcus pneumoniae, Escherichia coli, and thecatalytic activity order was consistent with no mutation of EPSP synthase, only thecatalytic activity of Agrobacterium CP4mutant EPSP synthase for PEP was improved,the others are decreased; The binding free energies of the six kinds of mutant EPSPsynthase with glyphosate were-6.94kcal·mol-1,-7.22kcal·mol-1,-6.72kcal·mol-1,-6.50kcal·mol-1,-6.62kcal·mol-1,-6.49kcal·mol-1, respectively, and the correspondinginhibition constants (Ki) were8.15?mol,5.06?mol,11.88?mol,17.34?mol,13.93?mol,17.55?mol, respectively, the Km/Ki values were6.29,4.65,3.45,1.26,2.14,2.30, respectively, and according to the Km/Ki values predicted six kinds of mutant EPSP synthase resistance to glyphosate from strong to weak as follows:Mycobacterium tuberculosis, Vibrio cholerae, Coxiella burnetii, Streptococcuspneumoniae, Agrobacterium CP4, Escherichia coli, and compared with no mutation,in addition to glyphosate resistance of CP4mutant EPSP synthase was weakened, theothers were enhanced, thus it can be estimated the Ala residue of in the active site hada great influence on the EPSP synthase resistance to glyphosate of six kinds oforganisms.