AlleyCat, a computationally designed Kemp eliminase produced by a single mutation in Calmodulin, a non-enzymatic metalloprotein, has been recently evolved by inducing several mutations around the substrate binding site into AlleyCat7. AlleyCat7 shows turnover numbers that are on par with those produced by state-of-the-art computational approaches. The traditional directed evolution process required seven rounds of mutagenesis and screening. In this work we describe a new technique that can be used as a novel strategy for protein design. Using a new approach, AlleyCat7 has been rapidly evolved into AlleyCat8 with a gain of more than two-fold in catalytic efficiency. Following the same strategy, this success story has been extended to myoglobin, a heme-binding protein which has been engineered to become a Kemp eliminase. NMR-guided evolution eliminates excessive mutagenesis and has the potential to unlock exciting avenues in directed evolution of protein catalysts.

NMR-guided directed evolution of Kemp eliminases

2019 Spring National Meeting

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