Semi-rational Engineering Of Methionine Adenosyl-transferase For Improved Catalytic Activity

S-adenosylmethionine（SAM）plays an important role in cell growth and is an important methyl donor in organisms.The enzymatic synthesis of SAM has the advantages of high stereoselectivity,mild reaction and easy separation and purification of the product.It is one of the important ways to synthesize SAM.By screening of Methionine adenosyl-transferase library,it was found that Methionine adenosyl-transferase Bf MAT from Bacteroides fragilis shows good product tolerance,but its catalytic activity is suitable for needs of industrial production.Therefore,Bf MAT was engineered by molecular transformation.The main research contents and results are as follows:（1）Screening mutants of Bf MAT（Methionine adenosyltransferase from Bacteroides fragilis,Bf MAT）.The protein structure of Bf MAT was built with Alpha Fold,and Discovery Studio was used for molecular docking.By analyzing the docking results,16 key sites of amino acids Val39,Ala40,Glu42,Glu55,Gln98,Ile102,Asp118,Thr267,Gly268,Ile271,Gly284,Gly285,Ala286,Lys290,Lys294 and Asp296 near substrate 5（?）were screened for saturation mutation.Through the screening of high-throughput method,the activity of single mutants E42M,E42R,E42C,E55L and K290I was improved.Then combined mutations were performed.It was found that E55L had synergistic effects with other mutants,while E42 and K290 had antagonistic effects with each other.The double mutants E42M/E55L,E42R/E55L,E42C/E55L and E55L/K290I with increased activity were obtained.At the same time,tribble mutants E42M/E55L/K290I,E42R/E55L/K290I and E42C/E55L/K290I were screened,and their activities were 2.13 times,2.10 times and 2.26 times that of WT,respectively.（2）Kinetic parameter comparison of Bf MAT.The optimal reaction p H of the mutants E42M/E55L/K290I,E42R/E55L/K290I and E42C/E55L/K290I decreased from 8.0 to 7.5relative to the optimal p H of WT.The kinetic parameters showed that the k_cat/K_m values of E42M/E55L/K290I and E42C/E55L/K290I were 4.22 and 7.81 times higher than that of WT,respectively,while the catalytic efficiency of E42R/E55L/K290I was not significantly improved because its K_m value was not significantly lower than that of WT.In 10 m M ATP and Met reaction,it was found that the good product tolerance of Bf MAT could make it suitable for long-time production.After 12 hours of reaction,the conversion rates of E42M/E55L/K290I,E42R/E55L/K290I and E42C/E55L/K290I were 30.40%,33.03%and 35.52%,improved11.4%,21.0%and 30.2%than WT.（3）Molecular docking and molecular dynamics simulations of Bf MAT.WT and mutants were analyzed by molecular docking and molecular dynamic simulation.Based on the molecular docking results,the reason for the improvement of the mutant catalytic efficiency is mainly due to the weakening of the electrostatic interactions between ATP and the substrate pocket,which can make ATP more conducive to enter the substrate pocket.The reason why the substrate affinity of mutant E42R/E55L/K290I is not significantly improved compared with the other mutations E42M/E55L/K290I and E42C/E55L/K290I may be due to the electrostatic attraction of positively charged of Arg to substrate ATP,it may make ATP unable to enter the substrate pocket with the correct conformation.Finally,we suggested that complex of E42M/E55L/K290I,E42R/E55L/K290I and E42C/E55L/K290I with ATP is more stable by analyzing short-range force and free energy.In conclusion,Bf MAT shows application and development potentialand its mutants show certain application value for industrial production.