Bottom-Up Nonempirical Approach To Reducing Search Space in Enzyme Design Guided by Catalytic Fields

[Image: see text] Currently developed protocols of theozyme design still lead to biocatalysts with much lower catalytic activity than enzymes existing in nature, and, so far, the only avenue of improvement was the in vitro laboratory-directed evolution (LDE) experiments. In this paper, we propose a different strategy based on “reversed” methodology of mutation prediction. Instead of common “top-down” approach, requiring numerous assumptions and vast computational effort, we argue for a “bottom-up” approach that is based on the catalytic fields derived directly from transition state and reactant complex wave functions. This enables direct one-step determination of the general quantitative angular characteristics of optimal catalytic site and simultaneously encompasses both the transition-state stabilization (TSS) and ground-state destabilization (GSD) effects. We further extend the static catalytic field approach by introducing a library of atomic multipoles for amino acid side-chain rotamers, which, together with the catalytic field, allow one to determine the optimal side-chain orientations of charged amino acids constituting the elusive structure of a preorganized catalytic environment. Obtained qualitative agreement with experimental LDE data for Kemp eliminase KE07 mutants validates the proposed procedure, yielding, in addition, a detailed insight into possible dynamic and epistatic effects.