A computational strategy to improve the activity of tyrosine phenol-lyase for the synthesis of L-DOPA.

Enzymes with high catalytic activity and stability are essential for industrial production, yet most natural enzymes do not meet these requirements. Therefore, efficient strategies for enzyme engineering are crucial. In this study, we developed a cost-effective computational design strategy to enhance the activity of tyrosine phenol-lyase (TPL) for the production of L-DOPA. By integrating structural analysis with computational design, and guided by our understanding of conformational flexibility of TPL, we identified a region where enhanced stability is most likely to facilitate enzyme activity. We screened stabilizing mutations by Cartesian_ddg in Rosetta. After identifying single stabilizing mutations, we grouped the nearby positions harboring multiple stabilizing mutations and calculated the energy of combinatorial variants. We found two promising groups where most variants exhibited lower calculated energy than the wild-type. Experimental validation showed five variants in these groups exhibit increased activity, with the two best variants showing catalytic activity enhancements of 1.8-fold and 1.6-fold compared to the wild-type enzyme.