An Artificial Metallolyase with Pliable 2-His-1-Carboxylate Facial Triad for Stereoselective Michael Addition

30 November 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


We repurposed the metal-binding site of a cupin superfamily protein into the 2-his-1-carboxylate facial triad, which is one of the common motifs in natural non-heme enzymes, to construct artificial metalloenzymes that can catalyze new-to-nature reactions. Cu2+-H52A/H58E variant catalyzed the stereoselective Michael addition reaction and was found to bear a flexible metal-binding site in the high-resolution crystal structure. Furthermore, the H52A/H58E/F104W mutant accommodated a water molecule, which was supported by Glu58 and Trp104 residues via hydrogen bonding, presumably leading to high stereoselectivity. Thus, the 2-his-1-carboxylate facial triad was confirmed to be a versatile and promising metal-binding motif for abiological and canonical biological reactions.


Artificial Metalloenzymes
Macromolecular Ligands
Protein Library
Cupin Superfamily
Copper Plasticity

Supplementary materials

SI Experimental, SI Tables and SI Figures


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