Manfred T. Reetz

Some time ago we developed a fundamentally new approach to asymmetric catalysis, specifically the directed evolution of enantioselective enzymes.¹ It is based on the appropriate combination of gene mutagenesis/expression and high-throughput ee-screening.² The organocatalytic enzymes that we have studied and are continuing to investigate are lipases, Baeyer-Villigerases and epoxide hydrolases. Highly enantioselective mutants have been evolved by the Darwinistic approach. QM/MM studies help to illuminate the source of enhanced enantioselectivity. In doing so we have used strategies for scanning protein sequence space which are based on error-prone PCR, saturation mutagenesis at the hot spots identified by epPCR and DNA shuffling. Although successful, these strategies are time-consuming. More recently, we have developed the Combinatorial Active-Site Saturation Test (CAST) and iterative CASTing, which we find particularly effective for expanding the substrate scope and enantioselectivity of enzymes.³ Iterative CASTing is an example of a more general concept, namely “iterative saturation mutagenesis”. Another example pertains to the drastic enhancement of protein stability as a result of applying this new form of directed evolution.⁴

1. First example: M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961-2963.; Angew. Chem., Int. Ed. Engl. 1997, 36, 2830-2832.
2. Comprehensive review: M. T. Reetz, Directed Evolution of Enantioselective Enzymes as Catalysts for Organic Synthesis. In: Advances in Catalysis, Vol. 49 (Eds.: B. C. Gates, K. Knözinger), Elsevier: San Diego, 2006, pp.1-69.
3. M. T. Reetz, M. Bocola, J. D. Carballeira, D. Zha, A. Vogel, Angew. Chem. 2005, 117, 4264-4268; Angew. Chem. Int. Ed. 2005, 44, 4192-4196. M. T. Reetz, L.-W. Wang, in part M. Bocola, Angew. Chem. 2006, 118, 1258-1263; Erratum, 2556; Angew. Chem. Int. Ed. 2006, 45, 1236-1241; Erratum, 2494. D. Belder, M. Ludwig, L.-W. Wang, M. T. Reetz, Angew. Chem. 2006, 118, 2523-2526; Angew. Chem. Int. Ed. 2006, 45, 2463-2466.
4. M. T. Reetz, J. D. Carballeira, A. Vogel, Angew. Chem. 2006, 118, 7909-7915; Angew. Chem. Int. Ed. 2006, 45, 7745-7751.