Kemp elimination catalyst

The 3D structure of a designed enzyme that acts as a Kemp elimination catalyst

(see also Directed evolution, 3iio, 3iip, and 3iiv)

A series of computationally designed enzymes that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has TIM barrel scaffold. The Kemp elimination of 5-nitrobenzisoxazole was chosen as a model reaction for proton (H) transfer from carbon, simultaneously with the cut of the nitrogen–oxygen (N-O) bond, resulting in cyanophenol product. Such reaction is a critical step in many enzymatic reactions. The catalytic base (E101), the general acid/H-bond donor (K222), and the stacking residue (W50) make interactions with the 5-nitrobenzisoxazole at the active site of KE07.

We can take a look at a comparison of the designed and crystal structures. The unbound crystal structure (lime) shows only limited structural changes of the active site side chains in comparison to the designed structure (red) modelled in the presence of the transition state analogue (yellow). Backbone RMSD for the active site is 0.32 Å versus 0.95 Å for the active site including the side chains. Overlap of the wildtype (lime) and Ile7Asp catalytically improved directed evolutionary mutant (blue-violet) structures. Directed evolution can significantly improve the stability, expression and activity of enzymes. Currently, it is the most widely used and successful strategy for biocatalysts' refinement. The hydrophobic residue Ile7 at the bottom of the active site was frequently mutated to polar or charged residues (the most common mutation is Ile7Asp), which holds Lys222 in position to stabilize the developing negative charge in the transition state while preventing interaction of Lys222 with Glu101. Indeed, the pK_a of the catalytic Glu101 shifts from <4.5 to 5.9 in the evolved variant with the Ile7Asp mutation.