Abstract
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.
Highlights
In recent years, intense research has been conducted in pursuit of harnessing the versatile protein capabilites for application in industrial biocatalysis.The major challenge arises from the fact that living organisms produce enzymes tailored for sustaining their own existence, rather than the needs of human society
The mutants obtained from laboratory-directed evolution (LDE) experiments differ mainly by the number of charged amino acids located in the second coordination sphere,[13,14] which may change conformation of its side chain upon docking with the substrate or nearby mutation.[34,35]
Speaking, the “intrinsic” contribution is a barrier calculated for reaction path coordinates as they are in catalysts, but without inclusion of molecular environment in quantum mechanics (QM) calculation
Summary
Intense research has been conducted in pursuit of harnessing the versatile protein capabilites for application in industrial biocatalysis. The mutants obtained from LDE experiments differ mainly by the number of charged amino acids located in the second coordination sphere,[13,14] which may change conformation of its side chain upon docking with the substrate or nearby mutation.[34,35] The importance of considering amino acid sidechain rotamers in biocatalysis has been recently demonstrated for Kemp eliminase[36] and triosephosphate isomerase mutants.[37] A more general catalytic field technique,[25,26] considering simultaneously both TSS8,9 and ground-state destabilization,[23] combined with the atomic multipole rotamer library scan, allows one to obtain valuable insight into active site preorganization This contribution constitutes the first attempt to test and validate the catalytic field technique and apply it to explore possible mutation nonadditivity, as well as dynamic and epistatic effects in biocatalysis resulting from side-chain rotations
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