Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.

BackgroundRecent years have seen enormous progress in the development of methods for modeling (bio)molecular systems. This has allowed for the simulation of ever larger and more complex systems. However, as such complexity increases, the requirements needed for these models to be accurate and physically meaningful become more and more difficult to fulfill. The use of simplified models to describe complex biological systems has long been shown to be an effective way to overcome some of the limitations associated with this computational cost in a rational way.Scope of reviewHybrid QM/MM approaches have rapidly become one of the most popular computational tools for studying chemical reactivity in biomolecular systems. However, the high cost involved in performing high-level QM calculations has limited the applicability of these approaches when calculating free energies of chemical processes. In this review, we present some of the advances in using reference potentials and mean field approximations to accelerate high-level QM/MM calculations. We present illustrative applications of these approaches and discuss challenges and future perspectives for the field.Major conclusionsThe use of physically-based simplifications has shown to effectively reduce the cost of high-level QM/MM calculations. In particular, lower-level reference potentials enable one to reduce the cost of expensive free energy calculations, thus expanding the scope of problems that can be addressed.General significanceAs was already demonstrated 40 years ago, the usage of simplified models still allows one to obtain cutting edge results with substantially reduced computational cost. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Abstract:The role of metal ions in catalyzing phosphate ester hydrolysis has been the subject of much debate, both in terms of whether they change the transition state structure or mechanistic pathway. Understanding the impact of metal ions on these biologically critical reactions is central to improving our understanding of the role of metal ions in the numerous enzymes that facilitate them. In the present study, we have performed density functional theory studies of the mechanisms of methyl triphosphate and acetyl phosphate hydrolysis in aqueous solution to explore the competition between solvent-and substrate-assisted pathways, and examined the impact of Mg 2 + on the energetics and transition state geometries. In both cases, we observe a clear preference for a more dissociative solvent-assisted transition state, which is not significantly changed by coordination of Mg 2 + . The effect of Mg 2 + on the transition state geometries for the two pathways is minimal. While our calculations cannot rule out a substrate-assisted pathway as a possible solution for biological phosphate hydrolysis, they demonstrate that a significantly higher energy barrier needs to be overcome in the enzymatic reaction for this to be an energetically viable reaction pathway.