A preorganization oriented computational method for de novo design of Kemp elimination enzymes

A preorganization oriented computational strategy for de novo enzyme design based on computational enzyme design tool PRODA was developed and demonstrated by the creation of Kemp elimination enzymes. A preorganized active site model of proton transfer from carbon with a low energy barrier was proposed and then anchored into the scaffold 3AOF, the endoglucanase from Thermotoga maritima, which was selected from the protein structural database. The low-energy amino acid sequences at the binding pocket to stabilize the catalytic productive geometry were computationally generated via the iterative protein redesign and molecular dynamics simulation. The designed variant (3AOF-KE03) bearing 17 mutations was experimentally confirmed to afford catalytic activity (kcat/KM = 14.04 M- 1s- 1) towards Kemp elimination, with measured rate (kcat = 0.033 s-1) enhancement of up to 104-fold. This computational strategy is general, and we anticipate the creation of a wide range of artificial enzymes to catalyze reactions with industrial significance in the future.