Design, Optimization and Characterization of a de novo Gold Hydroaminase

Artificial metalloenzymes (ArMs) offer a versatile and evolvable platform to extend the biocatalytic repertoire. Here we report the assembly of an ArM resulting from supramolecular anchoring an N-heterocyclic carbene Au(I) complex into a de novo designed tandem repeat protein (TRP). We identified a variant that, compared to the free cofactor, led to a higher catalytic activity for the Au-catalyzed hydroamination of 2-ethynylaniline. Structure-guided mutagenesis of this variant improved the activity, resulting in a double mutant displaying up to fourfold higher catalytic rates than the original TRP. Biophysical and crystallographic analysis revealed distinct cofactor binding poses, with single mutations reshaping the active site and correlating with improved catalytic performance. Importantly, the TRP scaffold imparted robustness, preserving catalytic activity under acidic conditions, in the presence of organic cosolvent, and at elevated temperatures, where the free cofactor was deactivated. This work highlights the potential of de novo designed proteins to harbor non-natural metal cofactors and points to design principles for stabilizing sensitive catalysts under chaotropic conditions.