Figure 4.

Designed enzymes. (a) The Kemp eliminase reaction scheme. (b) Structure of designed Kemp eliminase prior to directed evolution (3nyd) with two alternative ligand conformations (cyan and purple). Electron density contoured to 1.5 e^–/ų as a dark blue mesh, with a lower contour shown as a cyan volume representation at 0.3 e^–/ų. (c) Structure of final designed Kemp eliminase (4BS0) highlighting the hydrogen bond network. Disordered water replaces the acetate of the previous structure. Electron density contoured to 2.65 e^–/ų as a dark blue mesh, with a lower contour shown as a cyan volume representation at 0.3 e^–/ų. Difference density contoured to 0.3 e^–/ų, colored green for positive and red for negative. Hydrogen bonds in hydrogen bond network drawn as dashed black lines. (d) Disorder of tryptophan residue 44 in final Kemp eliminase. The modeled alternative conformation deposited in the PDB is shown in dark green. Electron density contoured as in panel b. (e) Possible alternative conformation of catalytic Asp127 as detected by two positive difference peaks indicating alternative positions of the carboxylic oxygens that stabilize interactions with alternative water conformations. Electron density contoured as in panel c.