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April 2nd, 2025
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University of Ottawa
biochemistry
biorxiv

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

Beck, J.Open in Google Scholar•Smith, B. J.Open in Google Scholar•Zarifi, N.Open in Google Scholar•Freund, E.Open in Google Scholar•Chica, R. A.Open in Google Scholar•Hoecker, B.Open in Google Scholar

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 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. Experimental testing identified one active variant with catalytic efficiency comparable to previously reported Kemp eliminases, and mutational analyses validated the essential role of the designed catalytic residues. Sequence optimization of this variant improved solubility and stability, enabling X-ray structure determination, which confirmed the designed lid structure. This study reports the first enzymatically active de novo TIM barrel and establishes a platform for designing enzymes from minimal protein scaffolds.

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