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Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Univ Minnesota, Dept Biochem Mol Biol & Biophys, 1479 Gortner Ave, St Paul, MN 55108 USA.;Univ Minnesota, Biotechnol Inst, 1479 Gortner Ave, St Paul, MN 55108 USA..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-3190-1173
2017 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 48, p. 17533-17546Article in journal (Refereed) Published
Abstract [en]

Organophosphate hydrolases are proficient catalysts of the breakdown of neurotoxic organophosphates and have great potential as both biotherapeutics for treating acute organophosphate toxicity and as bioremediation agents. However, proficient organophosphatases such as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzing lactonase SsoPox are unable to hydrolyze bulkyorganophosphates with challenging leaving groups such as diisopropyl fluorophosphate (DFP) or venomous agent X, creating a major challenge for enzyme design. Curiously, despite their mutually exclusive substrate specificities, PON1 and diisopropyl fluorophosphatase (DFPase) have essentially identical active sites and tertiary structures. In the present work, we use empirical valence bond simulations to probe the catalytic mechanism of DFPase as well as temperature, pH, and mutational effects, demonstrating that DFPase and PON1 also likely utilize identical catalytic mechanisms to hydrolyze their respective substrates. However, detailed examination of both static structures and dynamical simulations demonstrates subtle but significant differences in the electrostatic properties and solvent penetration of the two active sites and, most critically, the role of residues that make no direct contact with either substrate in acting as "specificity switches" between the two enzymes. Specifically, we demonstrate that key residues that are structurally and functionally critical for the paraoxonase activity of PON1 prevent it from being able to hydrolyze DFP with its fluoride leaving group. These insights expand our understanding of the drivers of the evolution of divergent substrate specificity in enzymes with identical active sites and guide the future design of organophosphate hydrolases that hydrolyze compounds with challenging leaving groups.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC , 2017. Vol. 139, no 48, p. 17533-17546
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-340260DOI: 10.1021/jacs.7b09384ISI: 000417669000051PubMedID: 29113434OAI: oai:DiVA.org:uu-340260DiVA, id: diva2:1179137
Funder
The Royal Swedish Academy of SciencesKnut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 306474Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2018-01-31Bibliographically approved

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