The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.M.L.C. and C.J.J. gratefully acknowledge support from the Australian Research Council under their Discovery Projects program (DP130102144), and the award of a DECRA fellowship to C.J.J. (DE120102673). M.L.C. gratefully acknowledges generous allocations of supercomputing time on the National Facility of the Australian National Computational Infrastructure. S.C.L.K. thanks the European Research Council, which provided financial support under the European Community’s Seventh Framework Programme (FP7/2007–2013)/ERC Grant Agreement 306474, the Knut and Alice Wallenberg Foundation for a Wallenberg Academy Fellowship, and the Swedish National Infrastructure for Computing (SNIC) for the generous allocation of supercomputing resources