Organophosphate Hydrolysis by a Designed Metalloenzyme: Impact of Mutations Explained

The efficient design of novel enzymes has been attainable only by a combination of theoretical approaches and experimental refinement, suggesting inadequate performance of de novo design protocols. Based on the analysis of the evolutionary trajectory of a designed organophosphate hydrolase, this work aimed at developing and validating the improved theoretical models describing the catalytic activity of five enzyme variants (including wild-type as well as theoretically derived and experimentally refined enzymes) performing the hydrolysis of diethyl 7-hydroxycoumarinyl phosphate. The following aspects possibly important for enzyme design were addressed: the level of theory sufficient for a reliable description of enzyme-reactant interactions, the issue of ground state (GS) destabilization versus transition state (TS) stabilization, and the derivation of the proper side chain rotamers of amino acid residues. For enzyme variants analyzed herein, differential transition state stabilization (DTSS, i.e., preferential TS binding by an enzyme over the GS binding) calculated with a non-empirical model of the interaction energy (i.e., multipole electrostatic plus approximate dispersion terms, MED) displayed a superior performance in ranking the enzyme catalytic activity. The MED DTSS-based systematic rotamer refinement performed with an efficient scanning procedure and accounting for long-range interaction energy terms is an important step capable of unlocking the full potential impact of the given residue that could be otherwise overlooked with a conventional static approach featuring optimization to the nearby minimum. While TS stabilization is the main factor contributing to the increased catalytic activity of the de novo-designed variant studied in this work, directed evolution refinement appears to impact the catalytic activity of another enzyme variant analyzed herein via GS destabilization.