The 3D structure of a designed enzyme that acts as a Kemp elimination catalyst
(see also Directed evolution, 3iio, 3iip, and 3iiv)
Publication Abstract from PubMed
The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 105 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in kcat/Km (kcat/Km of 2,600 M-1s-1 and kcat/kuncat of >106). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
Kemp elimination catalysts by computational enzyme design., Rothlisberger D, Khersonsky O, Wollacott AM, Jiang L, DeChancie J, Betker J, Gallaher JL, Althoff EA, Zanghellini A, Dym O, Albeck S, Houk KN, Tawfik DS, Baker D, Nature. 2008 May 8;453(7192):190-5. Epub 2008 Mar 19. PMID:18354394
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
A series of computationally designed enzymes that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has . The Kemp elimination of 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 . 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 .
We can take a look at a comparison of the and structures. The unbound crystal structure (lime) shows only limited structural changes of the side chains in comparison to the designed structure (red) modelled in the presence of the transition state analogue (yellow). RMSD for the active site is 0.32 Å versus 0.95 Å for the active site including the . of the wildtype (lime) and 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 at the bottom of the active site was frequently mutated to polar or charged residues (the most common mutation is ), which holds in position to stabilize the developing negative charge in the transition state while preventing interaction of Lys222 with . Indeed, the pKa of the catalytic Glu101 shifts from <4.5 to 5.9 in the evolved variant with the mutation.
