Investigating The Reason For The Improvement Of Catalytic Activity Of The Nitrilase Mutant From Synechocystis Sp.PCC6803 Through MD Simulation

Chiral? amino acids are widely used in the synthesis of drugs and pharmaceutical intermediates due to various biological activities.Although they have a very important clinical role,the synthesis of these molecules through chemical methods is still suffer-ing from many drawbacks.Biocatalysis has great prospects in the synthesis of small organic molecules due to its highly catalytic efficiency,stronger spatial selectivity,and environmental friendliness.At present,many natural or artificially modified enzymes have been used to synthesize various organic small molecules,and a large number of enzyme synthesis methods have been used to synthesize chiral gamma amino acids or their precursors,such as lipase,transaminase,and ene-reductase,nitrilase,etc.Studies have shown that the nitrilase?Ss NIT?from Synechocystis sp.strain PCC 6803 can asymmetrically catalyze 3-substituted glutaronitrile to produce optically active 3-sub-stituted-4-cyanobutanoic acid,which can be further converted into?S?-Pregabalin and?R?-Baclofen.After mutating the residues located in its active site,a series of mutants with enhanced activity were obtained.Among them,the triple mutants P194A/I201A/F202V have the highest catalytic activity and excellent stereoselectivity,while the double mutant H141D/F202V was inactive completely.However,the struc-tural basis for these two distinct changes in activity remains unclear.Based on the experimental data,we hope to study the reasons at the molecular level by using theoretical methods such as molecular docking,molecular dynamics sim-ulation,and binding free energy.The results show that the residues of the triple mutant can form relatively stable hydrogen bonds with the substrate?the hydrogen bond exist-ence rate is greater than 1?,Gly195 is the main residue that forms hydrogen bonds;the hydrogen bonds between double mutant and ligand are less than triple mutant;and the double mutant H141D/F202V has almost no hydrogen bond.Analysis of the distance between the S atom?nucleophilic attacking atom?of the key catalytic residue Cys169and the active carbon atom(C₁₃)of the substrate shows that the distance between the triple mutant is significantly lower than that of the wild type and double mutants,and the shortening of the distance may be beneficial for the catalytic residue reacting with the substrate.At the same time,we investigated the dihedral angle S-C_?-C_?-N of Cys169and found that the dihedral angle of the triple mutant was maintained at-60°,while the wild-type and double mutants maintained at-180°.This angle change may sterically promote the proximity of the substrate C₁₃ and the S atom of the catalytic Cys169.Through the binding free energy,we found that the free energy of binding between the three mutants and the substrate is as high as-111.286k J·mol^-1,while the free energy of binding of the wild-type and double mutants is relatively low,being-86.383 and-88.916 k J·mol^-1,respectively.Generally,the greater the binding free energy is,the tighter the substrate bind to the enzyme,the more easily it enters into the active site of the enzyme,the more accessibly a reaction to proceed.Finally,sampling the free energy landscape map,we found that in the three mutants,residue His141 had a 102°flip com-pared to the wild type.This change may throw out the ligand and bring it closer to the catalytic residue Cys169.In addition,the conformation of the indole ring of residue Trp170 in the double and triple mutants has also undergone very obvious changes:in the triple mutant,the indole ring of Trp170 rotates down relative to the wild type to push the ligand closer to C169,and the benzene ring of the substrate formed a?-?stacking interaction with C169,which also makes the ligand close to the isopropyl group of Val202 to form a p-?stacking interaction.These two interactions may stabilize the benzene ring of the substrate.As a result,the distance between the sulfur atom of Cys169 and the C13 atom of the substrate remains relatively low.We have also studied the inhibitory effect of the product,and the results show that the wild-type product exhibits obvious product inhibition due to the higher affinity of the product molecule,which makes it difficult for the product molecule to dissociate from the active site.In the triple mutant,the affinity of the product molecule is not as good as that of the substrate,so the product molecule cannot easily compete with the substrate for the active site,and the triple mutant has higher activity.The reason for this difference is that in the wild type,residue His141 forms a strong?-?stacking and p-?stacking interaction with the product molecule,which makes the product molecule to bind to the active site very stably.In the three mutants,I201A and F202V were mutated into smaller residue,and the product molecule can easily form multiple hydrogen bonds with Gly195,which is beneficial to the release of the product molecule.In this article,some of our simulation results are consistent with experimental con-clusions.In addition,we can find some residues through simulation that had not been reported in the experiment.These residues may play an important role in the substrate catalysis process.They may be used to modify Ss NIT to further improve the catalytic efficiency of enzymes,and ultimately enable Ss NIT to be used for large-scale industrial production of chiral gamma amino acids and its derivatives.