Parallel Evolution of Dirhodium Artificial Metalloenzymes Creates Distinct Reaction Environments for Selective Carbene Transfer

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Artificial metalloenzymes (ArMs) combine the reactivity of transition metal catalysts with the selectivity and evolvability of protein scaffolds. Understanding different mechanisms by which scaffolds can impart selectivity would greatly facilitate ArM design efforts. In this study, we conducted parallel directed evolution prolyl oligopeptidase (POP)-based dirhodium ArMs toward two mechanistically distinct carbene insertion reactions, N-H and Si-H insertion, to study differences in their stereoselectivity and reaction specificity. Starting from a common parent enzyme, iterative mutagenesis and screening yielded variants 6-A and 4-I, which exhibit high activity and enantioselectivity for N-H and Si-H insertion, respectively, along with orthogonal reaction specificity. Structural and computational studies, including molecular dynamics simulations, reveal that directed evolution promotes the emergence of distinct cofactor binding modes within the POP active site, corresponding to a polar environment for N-H insertion and a more hydrophobic environment for Si-H insertion. Mutagenesis experiments validate the functional relevance of these environments and highlight differences in local and long-range interactions governing catalysis. These findings demonstrate that parallel evolution on mechanistically divergent reactions can drive the formation of distinct catalytic microenvironments within a single enzyme scaffold, providing new insights into secondary-sphere engineering and the design of functionally specialized ArMs.

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