Many powerful reactions, particularly those catalyzed by non-biological metals, are not found in nature, so systems that combine the reactivity of metal catalysts with the evolvability and specificity of enzymes are highly sought after. To expand the scope of reactions are developing new classes of artificial metalloenzymes (ArMs), hybrid constructs comprised of protein scaffolds and metal catalysts (Fig. 2A).  Optimization of ArMs using directed evolution is being used to produce highly active enzymes for in vitro and in vivo transition metal catalysis.  We are focusing on incorporating privileged transition metal catalysts into protein scaffolds to generate bioorthogonal variants of known reactions.  This is illustrated by our recent work on dirhodium tetracarboxylate and manganese terpyridine ArMs (Fig. 2B/C). We then hope to demonstrate that scaffolds can be used to augment the reactivity of metal catalysts in order to access new reactions not possible in the absence of the scaffold protein. Ultimately, these enzymes will be utilized in metabolic engineering efforts for the biosynthesis of natural product derivatives and even completely synthetic compounds.  Such an approach would greatly facilitate the synthesis of complex molecules and enable exciting collaborations to explore the biological activity of these compounds.

Fig. 2. A) General scheme for ArM formation, and B) Representative cofactors.

Fig. 3. Representative dirhodium ArM and ArM-catalyzed enantioselective cyclopropanation.

In parallel with our ArM design and evolution efforts, we are pursuing detailed mechanistic studies on the systems developed in our laboratory to date. These studies involve steady state kinetics, biophysical experiments (e.g. FRET, LC-MS/MS, NMR, UV/Vis, etc.), and computational simulation (MD, QM/MM in collaboration with the Roux group at Chicago). Beyond shedding light on the function of these complex and fascinating systems, these studies are intended to inform future design efforts aimed at improving ArM activity and selectivity for synthetic applications.