Dan Tawfik lab: Directed evolution: Difference between revisions

A series of [http://en.wikipedia.org/wiki/Protein_design computationally designed enzymes] that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has <scene name='3iio/Int/10'>TIM barrel scaffold</scene>. The Kemp elimination of <scene name='3iio/Int/4'>5-nitrobenzisoxazole</scene> 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 <scene name='3iio/Int/8'>cyanophenol product</scene>. 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 <scene name='3iio/Int/9'>active site of KE07</scene>. [http://en.wikipedia.org/wiki/Directed_evolution Directed evolution] can significantly improve the stability, expression and activity of enzymes. In the catalytically improved directed evolutionary variants of KE07 containing the <scene name='3iio/Ali/7'>Ile7Asp mutation</scene>, Asp7 breaks the Glu101–Lys222 salt bridge (for example [[3iiv]], chain A is shown).