Customizing the Structure of a Minimal TIM Barrel to Craft a De Novo Enzyme

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 architectures lack the structural elements necessary 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. Experimental testing identified one active variant with catalytic efficiency comparable to previously reported Kemp eliminases. Mutational analyses confirmed that the designed lid is essential for catalysis, while molecular dynamics simulations revealed the importance of lid flexibility for substrate binding. This study reports the first enzymatically active de novo TIM barrel and establishes a platform for designing enzymes from minimal protein scaffolds.