De novo Kemp eliminases used in this work. Kemp elimination (Figure ) is a simple reaction that has been used as a common benchmark in de novo enzyme engineering studies. As shown in Figure , Kemp elimination is a classical model reaction for base-catalyzed proton abstraction from carbon, although there are increasing examples in the literature of redox mediated Kemp elimination. Kemp elimination is, in principle, an "easy" reaction that requires a very simple catalytic machinery, i.e., a catalytic base to remove the proton. Yet, it is not straightforward to reach high levels of catalysis for Kemp elimination, since the transition state (Figure ) is geometrically and electronically similar to the substrate and, therefore, achieving preferential stabilization of the former is challenging. This is for example evidenced by the only modest contribution of an oxyanion hole to catalysis in the highly proficient HG3.17 designed Kemp eliminase, as demonstrated by Hilvert and coworkers. Several Kemp eliminases from our previous work are used in this work to test our protein design workflow. These are summarized in Table and described in more detail below. As a starting point, we recently 28 used a minimalist design strategy (Figure ) to generate in b-lactamase scaffolds a completely new active site capable of catalysing Kemp elimination. This design approach involved replacing a large hydrophobic residue (a tryptophan) with an aspartate, in such a way that both, a cavity potentially capable to bind the Kemp substrate and a catalytic base potentially capable of performing proton abstraction, were generated through a single mutation. For this very simple design approach to be successful, the protein scaffold targeted for de novo active site generation must be able to tolerate a destabilizing disruptive mutation. Furthermore, since the cavity produced upon mutation does not exactly match the shape of the substrate molecule, the scaffold must be conformationally flexible, at least in the region of the new active site, in order to allow substrate binding, while at the same time not so flexible and solvent exposed that the substrate is unable to remain in the active site. Interestingly, while we tested a large number of modern (extant) and ancestral b-lactamases as scaffolds for potential Kemp elimination activity, the approach was only successful on proteins encoded by reconstructed ancestral b-lactamases sequences, obtained through ancestral sequence reconstruction.