Engineering the protein dynamics of an ancestral luciferase

Abstract Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role evolution, starting from evolvable and thermostable ancestral protein Anc HLD-RLuc which catalyses both dehalogenase luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis challenged scaffold dynamics. Screening for activities reveals InDel mutations localized three distinct regions that lead to altered (based on crystallographic B-factors, hydrogen exchange, molecular simulations). An anisotropic network model highlights importance conformational flexibility a loop-helix fragment Renilla luciferases ligand binding. Transplantation this dynamic leads lower product inhibition highly stable glow-type bioluminescence. The success our approach suggests strategy comprising (i) constructing template, (ii) mapping functional by mutagenesis, (iii) transplantation features, can functionally innovative proteins.