NMR-Guided Directed Evolution of a Kemp Eliminase.

Directed evolution has emerged as a powerful tool for improving protein properties and imparting completely new functionalities onto existing proteins. Several computational methods have been successful in predicting potential hotspots for directed evolution. However, they rely heavily on prior structural and/or functional information. To address these fundamental limitations, we focused on Nuclear Magnetic Resonance (NMR) as a tool to rapidly evolve enzymes without structural characterization. AlleyCat, a computationally designed Kemp eliminase produced by introducing a single mutation on C-terminal domain of a non-enzymatic metalloprotein Calmodulin, has recently been evolved into AlleyCat7. This traditional directed evolution process required seven rounds of mutagenesis and screening. A retrospective analysis of the evolved variants of AlleyCat series showed a remarkable correlation between chemical shift perturbation and probability of finding a productive mutation in the vicinity of that residue. Using our novel approach, AlleyCat7 has been rapidly evolved into AlleyCat10 with an improvement of about four-fold in catalytic efficiency after three successive rounds of evolution. NMR-guided evolution strategy eliminates excessive mutagenesis and has the potential to unlock exciting avenues in directed evolution of protein catalysts.