Fig. 2.

Optimization of an artificial Diels-Alderase. (A) The computational design DA_20_00 was optimized by cassette mutagenesis of active site residues (green balls) and epPCR (yellow balls) to afford DA_20_20. Mutations are mapped onto the DA_20_00 structure (PDB ID code 3I1C). (B) Combining these mutations with a computationally designed helix-turn-helix lid element (cyan backbone), followed by epPCR of the entire protein (red balls) and then the lid element (blue balls) yielded the proficient Diels-Alderase CE20. Mutations are mapped onto the CE6 structure (PDB ID code 3U0S). The active sites containing manually docked substrates (black carbons) are shown as transparent gray surfaces.