ActiveSite Hydrophobicity and the Convergent Evolutionof Paraoxonase Activity in Structurally Divergent Enzymes: The Caseof Serum Paraoxonase 1

摘要

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 markedlymore pronounced in the organophosphatase activity than the lactonaseactivity. Finally, a detailed comparison of PON1 to other organophosphatasesdemonstrates that either a similar “gating loop” ora highly buried solvent-excluding active site is a common featureof these enzymes. We therefore posit that modulating the active sitehydrophobicity is a key element in facilitating the evolution of organophosphataseactivity. This provides a concrete feature that can be utilized inthe rational design of next-generation organophosphate hydrolasesthat are capable of selecting a specific reaction from a pool of viablesubstrates.