2025 AIChE Annual Meeting
De Novo Design of Metalloclickases
The rise of computational de novo design has shifted enzyme design toward creating catalysts with pre-organized active sites and reaction-specific geometries that rival or surpass evolved enzymes. Although several de novo designed bond-breaking enzymes exist, the design of enzymes that promote bond formation remains a grand challenge. Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) is fast and reliable for site-selective coupling, but the cytotoxicity of free copper limits biological applications. The metal-free alternative strain-promoted azide alkyne cycloaddition (SpAAC) is biocompatible yet limited by substrate/product stability and selectivity. Previous efforts have yielded a single metal-free clickase for the azide-alkyne cycloaddition reaction, but it exhibits limitations on substrate scope and catalytic efficiency. Here, we computationally design metalloclickases, de novo metalloenzymes for CuAAC, to combine copper’s rate and scope with cellular compatibility and stereocontrol. To generate a metalloclickase that was biocompatible and highly efficient, we first located the ideal CuAAC active site by density functional theory (DFT), targeting a catechol oxidase–inspired bimetallic Cu(I) geometry. Each backbone was generated through RFDiffusion2 and refined with iterative rounds of partial diffusion to improve structural quality and substrate accessibility. Sequences were then generated using LigandMPNN, with resulting structures minimized and scored using Rosetta FastRelax. Finally, AlphaFold3 was used to predict folding of the apo state and transition state complexes, allowing evaluation of structural integrity and catalytic preorganization. This process was repeated iteratively, which yielded several promising results with Cα-RMSD <= 2, PTM >= 0.8, and active site RMSD <= 1. Experimental validation is under way. This approach offers a promising route toward designing enzymes for bond-forming reactions and expands the frontier of de novo enzyme design.
