<i>De Novo</i> Design, Directed Evolution and Computational Study of Heme-Binding Helical Bundle Protein Catalysts for Biocatalytic Enantioselective Ge–H Insertion

<i>De novo</i> designed proteins offer a malleable platform for the development of stereoselective transformations not found in biochemistry. Here, we report the <i>de novo</i> design and directed evolution of helical bundle protein catalysts for enantioselective germylation through Ge-H insertion, a transformation not previously achieved by enzymatic catalysis. Comparative computational analysis revealed that, relative to Si-H insertion, the Ge-H insertion reaction proceeds through an earlier and more flexible transition state, introducing distinct challenges for stereocontrol. Using a fully <i>de novo</i> designed truncated four-helix bundle scaffold as the starting point, directed evolution afforded a quadruple mutant that catalyzes Ge-H insertion with high efficiency, enantioselectivity, and broad substrate scope. Molecular dynamics simulations indicated that beneficial mutations introduced from directed evolution enhanced active-site preorganization and modulated local backbone flexibility, contributing to improved transition-state complementarity with fine-tuned binding pocket size and more stable cofactor positioning regulated by hydrogen bonding interactions. These findings showcase the excellent potential for <i>de novo</i> proteins to achieve stereoselective transformations previously unknown to biocatalysts and underscore the importance of active-site remodeling of <i>de novo</i> protein scaffolds via directed evolution in achieving selective catalysis involving flexible transition states.