Introducing Catalysis into Non-Catalytic Protein Folds: How Nature Does It

Introducing catalytic function into non-catalytic protein scaffolds remains a major challenge in the field of enzyme design, limiting our ability to produce novel enzymatic tools for use in synthetic biology systems. Using ancestral protein reconstruction, we investigated how nature navigates this problem by characterising the evolutionary trajectory from a non-catalytic solute binding protein (SBP) to a catalytically active cyclohexadienyl dehydratase (CDT). Functional characterisation of extant homologs of CDT and reconstructed ancestral proteins revealed that CDT evolved from an arginine-binding protein via an intermediate of unknown function. The emergence and improvement of catalysis occurred over several steps. Initially, a reactive catalytic motif was established by the incorporation of a general acid into a desolvated environment. The active site cavity was then reshaped to improve enzyme-substrate complementarity and hydrogen-bonding networks were introduced to precisely position the catalytic residue and contribute to transition state stabilization. Finally, remote substitutions refined the structure of the active site and contributed to changes in the conformational landscape of the enzyme, reducing the sampling of non-catalytic states. Interestingly, the molecular processes that underlie the emergence and evolution of CDT activity by natural selection are reflected in current approaches to computational design and directed evolution of enzymes in the laboratory.