Customizing the Structure of Minimal TIM Barrels to Craft Efficient De Novo Enzymes

Abstract The TIM barrel is the most prevalent fold in natural enzymes, supporting efficient catalysis of diverse chemical reactions. While de novo TIM barrels have been successfully 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 pocket for enzymatic function. Starting from two de novo TIM barrels, we designed nine variants with distinct lids to form active sites for the Kemp elimination. Four designs showed measurable activity, with the most active reaching a catalytic efficiency of 21,000 M⁻¹ s⁻¹ at its optimal pH. A co-crystal structure of this variant bound to a transition-state analogue confirmed the accuracy of the designed lid and active site. Using the X-ray structure of a lower-activity variant (19 M⁻¹ s⁻¹), we applied ensemble-based design to optimize its active site, increasing catalytic efficiency by >1,600-fold to 32,000 M⁻¹ s⁻¹. These results demonstrate that de novo TIM barrels can be endowed with substrate binding pockets supporting efficient catalytic function, establishing a platform for building enzymes on demand from minimal protein scaffolds.