Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

David Blaha-Nelson,Shina Caroline Lynn Kamerlin,Moshe Ben-David,Dennis M Krüger,Klaudia Szeler

doi:10.1021/jacs.6b10801

David Blaha-Nelson, Shina Caroline Lynn Kamerlin + Show 3 more

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https://doi.org/10.1021/jacs.6b10801

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Abstract

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.

Highlights

The chemical reactions that make life possible are dependent on enzymes for their regulation
While the most direct way to probe the importance of the conformation of Y71 and the active site loop on catalysis would be through crystal structures of paraoxonase 1 (PON1) in both its substrate-free form and in complex with different inhibitors, enzyme−ligand complexes of PON1 are notoriously difficult to crystallize,[7,16] and we have explored the role of Y71 and active site plasticity indirectly through a combination of site-saturation mutagenesis of Y71 and screening for both lactonase activity toward thiobutyl-γ-butyric lactone (TBBL) and paraoxonase activity
Following from our initial molecular dynamics simulations of wild-type and mutant forms of RePON1, we have modeled the effect of mutating Y71 on the energetics of TBBL and paraoxon hydrolysis using the empirical valence bond (EVB) approach, which is an empirically based hybrid VB/MM approach that uses classical force fields to model chemical reactivity within a quantummechanical framework.[62]

Summary

Introduction

The chemical reactions that make life possible are dependent on enzymes for their regulation. We have used the catalytically promiscuous organophosphatase serum paraoxonase 1 (PON1)[4−8] as a model system to understand the structural origins of the selectivity and promiscuity of this enzyme and have performed comparisons to other structurally diverse organophosphatases in order to find the chemical commonalities that facilitate their organophosphatase activities. As many organophosphatases have evolved from enzymes that were originally lactonases and/or arylesterases (and retain these activities to varying degrees),[4,9−12] we have focused on the particular features necessary to facilitate lactone and ester hydrolysis that are common among these enzymes

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