Customizing the structure of minimal TIM barrels to craft efficient de novo enzymes

The TIM barrel is the most prevalent fold in natural enzymes, supporting efficient catalysis of diverse reactions. While de novo TIM barrels have been designed, their minimalistic architecture lacks structural elements essential for substrate binding and catalysis. Here, we present CANVAS, a computational workflow that introduces a structural lid into a minimal de novo TIM barrel to anchor catalytic residues and form an active site. Starting from two scaffolds, we designed nine variants with tailored lids for the Kemp elimination. Four showed measurable activity, with the most active reaching a catalytic efficiency of 21,000 M^-1 s^-1. A cocrystal structure with a transition-state analog confirmed the accuracy of the designed lid and active site. Using the structure of a lower-activity variant, we applied ensemble-based design, increasing catalytic efficiency >1,600-fold to 32,000 M^-1 s^-1. These results demonstrate that de novo TIM barrels can be endowed with efficient catalytic function, establishing a platform for building enzymes from minimal protein scaffolds.