From binding protein to enzyme: investigating the emergence of cyclohexadienyl dehydratase activity from non-catalytic ancestral proteins

BE Clifton1, JA Kaczmarski1, PD Carr1, N Tokuriki2 and CJ Jackson1

  1. Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia
  2. Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada

Introducing catalytic function into non-catalytic protein scaffolds remains a major challenge in the field of enzyme design. 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 reveal that CDT evolved from an arginine-binding protein via an intermediate of unknown function. The mutations required to introduce CDT activity into a non-catalytic ancestral protein were identified using site-directed mutagenesis and directed evolution. Crystal structures and molecular dynamics simulations highlight the structural basis for the emergence of catalysis in the SBP fold and indicate that a change in the conformational landscape of the proteins is likely to have contributed to the evolution of CDT activity.