It is also of interest that conformational selection in our Kemp eliminase enzyme is linked to the roughly additive effects of interactions generated by an extra polypeptide chain and by mutations predicted by the FuncLib server 21 at catalytic hotspots determined through NMR chemical shift perturbation. Remarkably, the residues at such catalytic hotspot positions do not bear side-chains at the active site, thus supporting the potential of the NMR approach to guide directed evolution by identifying useful positions linked to dynamic effects that would not be obvious in the enzyme 3D-structure. We note that this NMR-guided approach has been successfully coupled with saturation mutagenesis to repurpose a natural enzyme, myoglobin, as a redox-mediated Kemp eliminase. Here, we extend this approach to a de novo active site, replacing saturation mutagenesis with FuncLib analysis. We further demonstrate that, in cases where NMR analysis is not practical or accessible, hotspots can be effectively predicted using Key Interactions Finder (KIF) to identify residues involved in functionally important non-covalent interactions, and then filtering high scoring residues using Key Interaction Networks (KIN) to eliminate residues that are involved in highly evolutionarily conserved interactions as hotspots.