Understanding and ultimately utilizing the enormous catalytic power of enzymes is a grand challenge for chemical biology. We have collaborated with biologists to design new enzymes with functions different from those evolved by Nature.1 Our successes showed that we understand which catalytic groups will accelerate the rates of reactions and that quantum-mechanical calculations predict the correct positioning of these groups. However, our understanding is not sufficient to rationally tune the global architecture of the protein that ultimately determines catalysis in order to achieve efficiencies rivaling those of the natural enzymes. Instead, experimental directed evolution is able to reach several orders of magnitude of acceleration beyond that achieved by the designed protein. We have recently used microsecond molecular dynamics (MD) simulations to reveal how mutations produced by laboratory evolution alter the structure and organization of enzyme active sites, describing the fluctuations between active and inactive conformations normally concealed to crystallography and rigid-backbone enzyme design.2,3 Here we report how directed evolution fine tunes the delicate balance between the necessary active site organization and the required flexibility of the binding regions to allow diffusion of unnatural reactants into the active site, dramatically enhancing stereoselective catalysis. We describe two highly enantioselective enzymes that share a common feature: the dispositions of the protein and cofactor catalytic groups are highly conserved along the evolutionary pathways, but not the flexible pockets and loops that determine the successful entrance and correct positioning of the prochiral substrates. Microsecond molecular dynamics were crucial to reveal these features, especially since substrates or inhibitors could not be co-crystallized with any of the evolved variants. 1 Kiss, G.; Çelebi-Ölçüm, N.; Moretti, R.; Baker, D.; Houk, K. N. Angew. Chem., Int. Ed. 2013, 52, 5700–5725. 2 Jiménez-Osés, G.; Osuna, S.; Gao, X.; Sawaya, M. R.; Gilson, L.; Collier, S. J.; Huisman, G. W.; Yeates, T. O.; Tang, Y.; Houk, K. N. Nat. Chem. Biol. 2014, 10, 431−436. 3 Osuna, S.; Jiménez-Osés, G.; Noey, E. L.; Houk, K. N. Acc. Chem. Res. 2015, 48, 1080–1089.

Impact of directed evolution on flexible binding domains explained by molecular dynamics

251st National Meeting (2016)

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