The protein LmrR is a versatile scaffold to design artificial metalloenzymes. Here, we show that the M8D/A92E (DE) mutations that result in a much more efficient catalyst, destabilize the protein dimerization interface and alter the conformation landscape when binding metal cofactor and substrates are shown. These results highlight the intricate relations between protein, metal cofactor, and substrates in defining catalytic efficiency.

Artificial metalloenzymes (ArM) hold great potential for the sustainable catalysis of complex new-to-nature reactions. To efficiently improve the catalytic efficacy of ArMs, a rational approach is desirable, requiring detailed molecular insight into their conformational landscape. Lactococcal multidrug resistance regulator (LmrR) is a multipurpose ArM scaffold protein that, when bound to the Cu(II)-phenanthroline cofactor, catalyzes the Friedel–Crafts alkylation (FCA) of indoles. Previously, the M8D and A92E mutations are found to increase the efficiency of this reaction, but a molecular explanation has been lacking. The impact of these two activating mutations on the conformational landscape of LmrR in its apo, cofactor- and substrate-bound states is determined. The mutations cause a marked destabilization of the dimerization interface, resulting in a more open central hydrophobic cavity and a dynamic equilibrium between dimer and monomer LmrR is found. While mutant and wild-type have similar pocket conformation in the cofactor-bound state, the mutant shows a distinct interaction with the substrate. Our results suggest that increased retention of the catalytic cofactor and widened plasticity improve the activity of the mutant. Ultimately, these results help elucidating the intricate relationships between conformational dynamics of the protein scaffold, cofactor, and substrates that define catalytic activity.