Improving The Catalytic Performance Of S-adenosylmethionine Synthetase Through Molecular Modification

S-adenosylmethionine(SAM),also known as S-adenosyl-L-methionine,is an important metabolic intermediate in organisms.And it participates in more than 40 biochemical reactions through transmethylation,transsulfurylation and polyamino synthesis.In clinic,SAM has been successfully applied to human therapy,such as liver disorders,depression and osteoarthritis.With further clinical research,pharmacological effects of SAM to other diseases,including Alzheimer’s disease,migraine and tumors,are also confirmed.At present,most studies focus on the product of SAM through the extraction of yeast cells that accumulate SAM after being cultivated in the medium supplemented with L-methionine,which is the industrial production method of SAM.However,this method is still unsatisfied within a long fermentation period,low conversion and complicated extraction processes.In contrast,enzymatic synthesis,known for its high efficiency and simple extract processes,has the potential to solve these problems.S-adenosylmethionine synthetase(MAT)catalyzes the only enzymatic synthesis of SAM in cells from ATP and L-methionine,while producing by-product orthophosphoric acid(Pi)and pyrophosphate(PPi).In order to choose a suitable MAT for the production of SAM,this study selected 8 genes for the synthesis of MATs based on the articles about MATs and strains used in our laboratory for heterologous expression of MATs in Escherichia coli BL21(DE3)using pET-28a(+)as the vetors.Thus,a library of MATs was constructed.By comparing the cell growth and enzyme properties of the recombinant bacteria after induction,it was found that the MAT from E.coli(eMAT)showed better soluble expression and higher specific activity,so it was selected for further study.However,the specific activity of the wild-type eMAT was just 1.82 U/mg at 37℃.When the concentration of SAM was over 1 mM in the reaction system,the relative activity was less than 20%,which was unfavourable of the accumulation of SAM.Therefore,the catalytic activity of eMAT was engineered by molecular modification.Firstly,a high throughput screening method suitable for this study was constructed according to the spectrophotometric assay of Pi,and a mutant library was successfully constructed by optimizing the concentration of Mn2+ in error-prone PCR system.After continuous screening,a variant R74H was obtained,of which the specific activity increased to 4.15 U/mg when the specific activity of wild-type eMAT was 1.82 U/mg.At the same time,the wild-type eMAT was used as parent for site-directed mutagenesis based on the article about catalytic activities of MATs.Variant I303V and I65V were obtained and their specific activities were 2.76 U/mg and 4.57 U/mg,respectively.In order to test the feasibility of mutants to produce SAM,the concentrations of SAM at different concentrations of substrates were examined.The result showed that the SAM produced by wild-type eMAT was only 1.62 mM when the SAM produced by variant R74H,I65V and 1303V were 1.48 mM,1.84 mM and 2.40 mM in the present of 10 mM substrates.The Ki ATP and Ki,L-met of variant 1303V were 0.20 mM and 0.23 mM while Ki,ATP and Ki,L-met of variant R74H were 0.05 mM and 0.20 mM,which indicated that the product inhibition was the major factor that prevented the accumulation of SAM.Therefore,the variant 1303V with lower product inhibition was used as the parent for molecular modification to reduce product inhibition.Considering that the product inhibition of variant I65V was lower than wild-type eMAT,variant I303V/I65V was acquired by site-directed mutagenesis.The severe product inhibition of eMAT may be due to the fact that SAM prevents the binding of ATP to the enzyme.Since ATP was located in the highly conserved active site,semi-rational design of non-conservative sites near the active site was performed.The results indicated that not only the SAM concentration of variant I303V/I65V/L186V increased to 3.27 mM,but also the specific activity got a 1.54-fold increase.Though the variant I303V/I65V/N104A showed an increase in specific activity,the concentration of SAM decreased to less than 2 mM,so a site-saturated mutagenesis was performed on 104th of variant I303V/I65V/L186V.Through high throughput screening,the specific activity of variant I303V/I65V/L186V/N104K reached 6.02 U/mg,but the SAM produced by it decreased to 2.68 mM with 10 mM substrates.In addition,the optimum temperature of the variant I303V/I65V/L186V/N104K was 55℃ and its optimum pH was 8.0 while the optimum temperature of wild-type eMAT was 50℃ and optimum pH was 8.4.The enzymatic activity of the variant remained 80%after heat treatment at 37℃ for 8 h while the wild-type eMAT remained just 30%.In general,the catalytic performance of variant I65V/I303V/L186V/N104K obtained by molecular modification in this study was significantly improved when compared wild type eMAT and the reported MATs.