Kemp elimination catalyst

spinqualitylabelsall models Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D 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 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 pKa of the catalytic Glu101 shifts from <4.5 to 5.9 in the evolved variant with the Ile7Asp mutation.

About this Structure

2RKX is a Directed evolution structure. Full crystallographic information is available from OCA. </StructureSection>

Primary Reference

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

Created with the participation of Michal Harel, Jaime Prilusky, Eran Hodis.

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Alexander Berchansky