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Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1
journal contribution
posted on 2016-12-27, 00:00 authored by David Blaha-Nelson, Dennis M. Krüger, Klaudia Szeler, Moshe Ben-David, Shina Caroline Lynn KamerlinSerum
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.