Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes : The Case of Serum Paraoxonase 1
Blaha-Nelson, David ; Krüger, Dennis M ; Szeler, Klaudia ; Ben-David, Moshe and Kamerlin, Shina Caroline Lynn LU
(2017)
In Journal of the American Chemical Society
139(3).
p.1155-1167
- 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... (More)
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.
(Less)
- author
- Blaha-Nelson, David
; Krüger, Dennis M
; Szeler, Klaudia
; Ben-David, Moshe
and Kamerlin, Shina Caroline Lynn
LU
- publishing date
- 2017-01-25
- type
- Contribution to journal
- publication status
- published
- keywords
- Aryldialkylphosphatase/chemistry, Binding Sites, Biocatalysis, Humans, Hydrolysis, Hydrophobic and Hydrophilic Interactions, Lactones/chemistry, Molecular Dynamics Simulation, Mutation, Paraoxon/chemistry, Protein Conformation
- in
- Journal of the American Chemical Society
- volume
- 139
- issue
- 3
- pages
- 13 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:28026940
- scopus:85019116019
- ISSN
- 1520-5126
- DOI
- 10.1021/jacs.6b10801
- language
- English
- LU publication?
- no
- id
- c3e9959b-a22f-4753-baf1-0098c85f5b8c
- date added to LUP
- 2025-01-11 21:25:34
- date last changed
- 2025-06-29 18:15:12
@article{c3e9959b-a22f-4753-baf1-0098c85f5b8c,
abstract = {{<p>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.</p>}},
author = {{Blaha-Nelson, David and Krüger, Dennis M and Szeler, Klaudia and Ben-David, Moshe and Kamerlin, Shina Caroline Lynn}},
issn = {{1520-5126}},
keywords = {{Aryldialkylphosphatase/chemistry; Binding Sites; Biocatalysis; Humans; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Lactones/chemistry; Molecular Dynamics Simulation; Mutation; Paraoxon/chemistry; Protein Conformation}},
language = {{eng}},
month = {{01}},
number = {{3}},
pages = {{1155--1167}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Journal of the American Chemical Society}},
title = {{Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes : The Case of Serum Paraoxonase 1}},
url = {{http://dx.doi.org/10.1021/jacs.6b10801}},
doi = {{10.1021/jacs.6b10801}},
volume = {{139}},
year = {{2017}},
}