Effect of Binding on Enantioselectivity of Epoxide Hydrolase

Abstract

Molecular dynamics simulations and free energy calculations have been used to investigate the effect of ligand binding on the enantioselectivity of an epoxide hydrolase (EH) from Aspergillus niger. Despite sharing a common mechanism, a wide range of alternative mechanisms have been proposed to explain the origin of enantiomeric selectivity in EHs. By comparing the interactions of ( R)- and ( S)-glycidyl phenyl ether (GPE) with both the wild type (WT, E = 3) and a mutant showing enhanced enantioselectivity to GPE (LW202, E = 193), we have examined whether enantioselectivity is due to differences in the binding pose, the affinity for the ( R)- or ( S)- enantiomers, or a kinetic effect. The two enantiomers were easily accommodated within the binding pockets of the WT enzyme and LW202. Free energy calculations suggested that neither enzyme had a preference for a given enantiomer. The two substrates sampled a wide variety of conformations in the simulations with the sterically hindered and unhindered carbon atoms of the GPE epoxide ring both coming in close proximity to the nucleophilic aspartic acid residue. This suggests that alternative pathways could lead to the formation of a ( S)- and ( R)-diol product. Together, the calculations suggest that the enantioselectivity is due to kinetic rather than thermodynamic effects and that the assumption that one substrate results in one product when interpreting the available experimental data and deriving E-values may be inappropriate in the case of EHs.

Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

“…Experimental data produced from studies of protein-ligand interactions is inherently uncertain: three-dimensional crystal structures of proteins (generated from X-ray, NMR and neutron diffraction) are generally average structures that do not provide information on the dynamics of proteinligand complexes and properties that promote improved reaction conditions, experiments typically produce values averaged over both time and molecular space that are scarce relative to the number of degrees of freedom involved [72], and measurements of enzyme selectivity may be inaccurate due to errors from poor sample manipulation [73] or incorrect assumptions regarding the underlying reaction mechanism [24]. To reduce this uncertainty and assist in the interpretation of experimental data, molecular modelling and simulation methods such as protein-ligand docking, molecular dynamics and quantum mechanics are used to describe protein-ligand systems in atomic detail [72].…”

“…To reduce this uncertainty and assist in the interpretation of experimental data, molecular modelling and simulation methods such as protein-ligand docking, molecular dynamics and quantum mechanics are used to describe protein-ligand systems in atomic detail [72]. These methods have been used extensively to provide insight into the selective properties of a number of EHs [24,31,32,74,75] and are applied in Chapters 6 and 7 to better understand the selectivity of AnEH and its mutants.…”

“…EHs are ubiquitous in nature [15] and are part of the α/β -hydrolase super-family, whose members are characterised by a shared structural fold and catalytic triad [13]. A wealth of structural and biochemical data has been generated for EHs [1,[16][17][18][19][20][21][22][23], however the origin(s) of their enantio-and regioselective (Figure 1.1) properties remains uncertain [24]. EHs therefore continue to be actively studied in order to better understand, predict and improve their selectivities [24][25][26][27][28][29][30][31][32].…”