Understanding Complex Mechanisms of Enzyme Reactivity: The Case of Limonene-1,2-Epoxide Hydrolases

Abstract

Limonene-1,2-epoxide hydrolases (LEHs), a subset of the epoxide hydrolase family, present interesting opportunities for the mild, regio-and stereo-selective hydrolysis of epoxide substrates. However, moderate enantioselectivity for non-natural ligands, combined with narrow substrate specificity, has so far limited the use of LEHs as general biocatalytic tools. A detailed molecular understanding of the structural and dynamic determinants of activity may complement directed evolution approaches to expand the range of applicability of these enzymes. Herein, we have combined quantum mechanics/molecular mechanics (QM/MM) free energy calculations for the reaction with MD simulations of the enzyme internal dynamics, and the calculation of binding affinities (using the WaterSwap method) for various representatives of the enzyme conformational ensemble, to show that the presence of natural or non-natural substrates differentially modulates the dynamic and catalytic behavior of LEH. The cross-talk between the protein and the ligands favors the selection of specific substrate-dependent interactions in the binding site, priming reactive complexes to select different preferential reaction pathways. The knowledge gained via our combined approach provides a molecular rationale for LEH substrate preferences. The comprehensive strategy we present here is general and broadly applicable to other cases of enzyme-substrate selectivity and reactivity.

Molecular Dynamics Simulation Framework to Probe the Binding Hypothesis of CYP3A4 Inhibitors

“…The WaterSwap method uses explicit water molecules [76], so does not suffer from the same limitations as the MM(GB/PB)SA method. The WaterSwap method has been applied successfully to other systems [77,78,79,80,81,82,83,84]. Herein, we apply the WaterSwap method to estimate the binding free energies for the five highly potent inhibitors of CYP3A4.…”

Tuning Proton Transfer Thermodynamics in SARS-Cov-2 Main Protease: Implications for Catalysis and Inhibitor Design

“…The hybrid quantum/classical approach we apply here, the MD-PMM approach, 11,12 is based on the joint use of classical MD simulations and quantum mechanical (QM) calcula-tions. As commonly done in hybrid multiscale approaches, [13][14][15][16] also for the investigation of enzyme catalysis, 5,7,[17][18][19][20][21] the portion of the system in which the chemical event takes place is treated quantum mechanically (the quantum center, QC) while the rest of the system is treated classically and atomistically and exerts an electrostatic perturbation on the QC electronic states. However, the main difference with other hybrid methods is that in the MD-PMM the whole system configurational space (including the QC) is sampled by fully classical MD simulations.…”

Structure-function investigation of 3-methylaspartate ammonia lyase reveals substrate molecular determinants for the deamination reaction

“…Moreover, we cannot simulate association and dissociation between the enzyme and the ligands and account for the reactivity. Such studies will require a long-term approach and exhaustive calculations with a plethora of approaches [34][35][36][37], for which this study has laid the foundations.…”