Cloning,Screening And Rational Design Of O-Methyltransferase

O-methylation reaction is used for methylation modification of compounds,production of aromatic ethers,and protection of carboxyl group,and thus has wide applications in chemical industry.Since the traditional methylating reagents are featured by high toxicity and low structural specificity,an alternative green methylation process is required.O-methyltransferase is the specific biocatalyst for O-methylation reaction,with high selectivity and mild reaction conditions,which has great potential in industrial applications.Moreover,O-methyltransferase has been identified as the rate-limting enzyme in the biosynthesis of melatonin and coenzyme Q10,causing the major metabolic bottleneck.Therefore,mining and engineering of O-methyltransferase is of great significance.In this paper,an O-methyltransferase library was constructed by gene mining.By screening with multiple substrates,AtCOMT(Arabidopsis thaliana Caffeic acid O-methyltransferase)with broad substrate spectrum was selected out for further study.Considering its potential application in melatonin biosynthesis,the activity of AtCOMT in NAS(N-acetylserotonin)O-methylation was improved by rational design,and the molecular mechanism behind the activity improvement was illustrated by kinetic analysis and molecular dynamic simulations.In more details,this paper studied the following issuess:(1)Diversified O-methyltransferases were selected by gene mining,followed by cloning and expression in E.coli BL21.Sixteen O-methyltransferases were successfully cloned and expressed,constructing an O-methyltransferase library.(2)Various valuable and available O-methylation reactions were used to screen the O-methyltransferases,and the corresponding analytical methods were developed by HPLC or GC.The O-methyltransferase AtCOMT with broad substrate spectrum was selected for further study.(3)On account that AtCOMT was able to catalyze the conversion of NAS to melatonin,which had been reported as the major bottleneck in melatonin biosynthesis,AtCOMT was engineered for enhanced activity in NAS O-methylation.Based on the structure of NAS-AtCOMT complex constructed by homologous modeling and molecular docking,a rational design strategy was proposed to enhance the interactions between the substrate-binding pocket and NAS.Finally,a triple mutant C296F-Q310L-V314T with 8.5-fold higher activity than the wild type was obtained.(4)The kinetic analysis data and molecular dynamic simulations of the mutant in comparison with the wild-type AtCOMT showed that the affinity and catalytic efficiency of the mutant to NAS were both imrpoved and suggested the occurrence of synergistic effect among mutation sites.The binding of NAS with Phe296 and Leu310 was enhanced,and Thr314 enhanced NAS binding by affecting the protein structure in its surrounding.In summary,the catalytic activity of the triple mutant C296F-Q310L-V314T was increased by enhancing the binding between the enzyme and NAS.