| It is the main purpose of this thesis to demonstrate that the technique of protein design, which has been used in the past decade to successfully redesign protein cores, protein-protein, protein-DNA and protein-small molecule interfaces, can be extended to the design of new enzymes de novo. Once formulated in its generality, the problem of enzyme design is separated into three steps: (i) generation of an ideal active site that will maximally stabilize the highest-activation energy transition-state along the reaction pathway for the reaction to be catalyzed, (ii) placement of such a minimal active site into a set of scaffolds so that the transition structure fits into the scaffolds and the catalytic sidechain originates from the protein scaffolds and (iii) design of the remaining of the active site pocket, i.e. changing the sequence of the pocket as to further stabilize the placed transition structure and the positioning of the catalytic residues. The algorithms developed have been benchmarked against a set of 10 naturally occurring enzyme for which a high-resolution crystal structure is available and were able to recapitulate the native active site in 80% of the cases.;This methodology has been successfully applied to the design of enzymes catalyzing a Diels-Alder reaction between a carbamate-substituted butadiene and N,N-dimethyl acrylamide. The designs have been carried out against the endo transition state. One active design was obtained, and its activity monitored by LC/MS/MS. Further semi-rational library and screening led to the identification of two other variants with increased activity. The most active design shows a kcatKM1KM2k uncat exceeding 106M-1 while its rate enhancement in saturating dienophile condition is 10 times higher than the best existing protein catalysts for this reaction. In addition, the design is more than 95% specific for the endo adduct. Finally, up to 8 turn-overs were measured. This represents the first account of a computationally designed stereoselective bimolecular reaction. |
