Crystal structure of Renilla reniformis luciferase variant RLuc8-W121F/E144Q in complex with a coelenteramide (the postcatalytic enzyme-product complex)
Schenkmayerova, A., Damborsky, J., Marek, M. Luciferase from Renilla reniformis (RLuc) catalyzes the degradation of coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light (EC 1.13.12.5). This enzyme belongs to the Haloalkane dehalogenase family II with a different catalytic function (EC 3.8.1.5) Reconstruction of an ancestral enzyme shows it has both hydrolase and monooxygenase activities ( Chaloupkova et al.) Preprint available of: Engineering Protein Dynamics of Ancestral Luciferase DOI: 10.26434/chemrxiv.12808295
Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein Anc(HLD-RLuc) which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of Anc(HLD-RLuc) challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.
Insertion-deletion mutations are sources of major functional innovations in naturally evolved proteins, but directed evolution methods rely primarily on substitutions. Here, we report a powerful strategy for engineering backbone dynamics based on InDel mutagenesis of a stable and evolvable template, and its validation in application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive multidisciplinary analysis linked the conformational flexibility of a loop-helix fragment to binding of the bulky substrate coelenterazine. The fragment's key role in extant Renilla luciferase was confirmed by transplanting it into the ancestor. This increased its catalytic efficiency 7,000-fold, and fragment-containing mutants showed highly stable glow-type bioluminescence with 100-fold longer half-lives than the flash-type Renilla luciferase RLuc8, thereby addressing a limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a robust template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of a dynamic feature, provides a potent strategy for discovering protein modifications with globally disruptive but functionally innovative effects.
Representative scheme of Haloalkane_dehalogenase-HLD2 structure and an image from PDBsum server
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