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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: 2018-09-14Bibliographically approved
In thesis
1. Computational Modeling of the Mechanisms and Selectivity of Organophosphate Hydrolases
Open this publication in new window or tab >>Computational Modeling of the Mechanisms and Selectivity of Organophosphate Hydrolases
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Computational modeling is becoming an increasingly integral part of (bio)chemistry, providing a powerful complementary view into the dynamics, binding, and reactivity of biochemical systems. In particular, molecular simulations based on multiscale models are now regularly employed in studies of enzymatic reactions, offering invaluable mechanistic insight through the lens of molecular energy landscapes. In this thesis, I used the empirical valence bond (EVB) and related methods to study the mechanisms and selectivity of organophosphate hydrolases.

Organophosphate hydrolases are a diverse class of enzymes capable of degrading some of the most toxic compounds known to mankind, including pesticides and chemical warfare agents. They are particularly interesting from a mechanistic and evolutionary point of view, having evolved the ability to catalyze the hydrolysis of compounds which were introduced to nature less than a century ago. Moreover, they show promise as effective organophosphate decontamination agents and a thorough understanding of their function is fundamental to the future design of efficient and selective biocatalysts. 

As organophosphate hydrolases are metal-dependent enzymes, a reliable metal model was a prerequisite to our simulations. First, I present the development of force-field independent parameters for several alkaline-earth and transition-metal ions described using the nonbonded cationic dummy model. The model was subsequently employed in EVB simulations to probe the origin of metal-ion activity and selectivity patterns observed in methyl parathion hydrolase (MPH) and to provide mechanistic insight into its paraoxonase and promiscuous arylesterase activities. I further set out to resolve open mechanistic questions surrounding diisopropyl fluorophosphatase (DFPase) by performing extensive simulations of two mechanistic pathways proposed in literature, including calculating the effects of mutations, temperature, and protonation states on the rate of hydrolysis. Using this knowledge, I address the origin of cross-selectivity between DFPase and a structurally similar enzyme serum paraoxonase 1 (PON1). Finally, I present the latest developments in the software used to perform the simulations.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 92
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1722
Keywords
Organophosphate Hydrolase, Computational enzymology, MPH, DFPase, PON1, Empirical Valence Bond, EVB
National Category
Biochemistry and Molecular Biology Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-360518 (URN)978-91-513-0444-1 (ISBN)
Public defence
2018-11-02, BMC:B22, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-10-08 Created: 2018-09-14 Last updated: 2018-10-16

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