Molecular Modification And Study On Structure-activity Relationship Of Tyrosinase From Bacillus Aryabhattai

L-DOPA is one of the most effective drugs for the treatment of Parkinson’s disease.With the increasing incidence of Parkinson’s disease and the development of new applications for L-DOPA,the demand for L-DOPA is increasing year by year.At present,the production of L-DOPA mainly relies on chemical synthesis,but there are several problems including complex reaction process,high cost of metal catalyst,and so on.Enzyme catalytic method has become a new approach for the production of L-DOPA,due to the advantages of strong substrate specificity and high catalytic efficiency.It is promising to synthesis L-DOPA by using tyrosinase to catalyze tyrosine.However,at present,the monophenolase activity of most natural tyrosinase are relatively low,and with poor stability,which cannot be used for the efficiently production of L-DOPA.In order to improve the monophenolase activity of tyrosinase,in this study,a tyrosinase from Bacillus aryabhattai（ab TYR）was selected for iterative saturation mutation and screening for mutants with high monophenolase activity.Firstly,homology modeling and molecular docking analysis were performed for ab TYR.The catalytic activity of tyrosinase belongs to the six conserved histidine residues and coordinate two copper ions,so,the hotspot domain of copper ion binding site was selected as the first mutation domain.According to the docking results,the substance of tyrosine interacted with several residues after entering the binding pocket of ab TYR,thus,the substrate binding sites hotspot domain was selected as the second mutation domain.In the process of tyrosine catalysis by tyrosinase,a conserved water molecule is always existed to mediate the deprotonation of the substrate,therefore,the water molecule binding sites hotspot domain was selected as the third mutation domain.The mutant of MT6（G43R/M61H/A232C/Q214D/V217A/F197W）with high monophenolase activity was screened by iterative saturation mutagenesis strategy.The specific activity of MT6 was 4.17-fold of WT,K_m was 3.04-fold of WT,K_cat was 3.88-fold of WT,and the catalytic efficiency(K_cat/K_m)was 11.79-fold of WT.The inter-molecular interaction between the ab TYR and tyrosine before and after mutation was furtherly analyzed by molecular dynamics simulation.After the mutation,tyrosine changed its position in the substrate binding site domain of ab TYR,and generated an additional hydrogen bond with the ab TYR,which made the interaction between tyrosine and ab TYR stronger.In addition,the mutation of amino acid sites caused MT6 mutant to form a larger substrate binding site space than WT,which was easier for tyrosine to bind to ab TYR.Moreover,the MT6 mutant had a more stable structure than WT,beneficial for the stability of ab TYR.The generation of L-DOPA was catalyzed using the MT6 mutant with tyrosine as substrate,the final L-DOPA yield was 84%.