Enhanced chemoselectivity of a plant cytochrome P450 through protein engineering of surface and catalytic residues.

Cytochrome P450s (P450s) are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites. Given the high degree of gene redundancy and challenge to functionally characterize plant P450s, protein engineering is used as a complementary strategy to study the mechanisms of P450-mediated reactions, or to alter their functions. We previously proposed an approach of engineering plant P450s based on combining high-accuracy homology models generated by Rosetta combined with data-driven design using evolutionary information of these enzymes. With this strategy, we repurposed a multi-functional P450 (CYP87D20) into a monooxygenase after redesigning its active site. Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells, expressing them in heterologous hosts usually results in problems of expression or activity. Here, we applied computational design to tackle these issues by simultaneous optimization of the protein surface and active site. After screening 17 variants, effective substitutions of surface residues were observed to improve both expression and activity of CYP87D20. In addition, the identified substitutions were additive and by combining them a highly efficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis. This study shows the importance of considering the interplay between surface and active site residues for P450 engineering. Our integrated strategy also opens an avenue to create more tailoring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol, the precursor of natural sweetener mogrosides. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00056-z.