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Computational modelling of enzyme selectivity
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enantioselective reactions are one of the ways to produce pure chiral compounds. Understanding the basis of this selectivity makes it possible to guide enzyme design towards more efficient catalysts. One approach to study enzymes involved in chiral chemistry is through the use of computational models that are able to simulate the chemical reaction taking place. The potato epoxide hydrolase is one enzyme that is known to be both highly enantioselective, while still being robust upon mutation of residues to change substrate scope. The enzyme was used to investigate the epoxide hydrolysis mechanism for a number of different substrates, using the EVB approach to the reaction both in solution and in several enzyme variants. In addition to this, work has been performed on new ways of performing simulations of divalent transition metals, as well as development of new simulation software.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 104
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1530
Keywords [en]
enantiomer, epoxide hydrolase, chiral catalysis, empirical valence bond approach, method development
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-326108ISBN: 978-91-513-0005-4 (print)OAI: oai:DiVA.org:uu-326108DiVA, id: diva2:1118787
Public defence
2017-09-13, A1:111 BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-08-21 Created: 2017-07-02 Last updated: 2018-12-03
List of papers
1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
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2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
Abstract [en]

The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
Funder
Swedish National Infrastructure for Computing (SNIC), 2013/26-1
Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2018-12-03Bibliographically approved
2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
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2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
Abstract [en]

Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
Funder
EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
Abstract [en]

Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
4. Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
Open this publication in new window or tab >>Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
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2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed) Published
Abstract [en]

Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only due to changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. To probe the structural origin of these differences in substrate selectivities and catalytic regiopreferences, we have solved the crystal structures for the in-vitro evolved StEH1 variants. We have additionally used these structures as a starting point for docking the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking calculations, the apparent preferred binding modes obtained from the docking appears to rationalize the experimentally determined regioselectivities. These calculations could also identify an active site residue (F33) as a putatively important interaction partner, a role that could explain the high degree of conservation of this residue during evolution. Overall, our combined experimental, structural and computational studies of this system provide snapshots into the evolution of enantioconvergence in StEH1 catalyzed epoxide hydrolysis.

Keywords
enantioselectivity; epoxide hydrolysis; evolutionary snapshots; laboratory evolution; protein engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-298675 (URN)10.1002/cbic.201600330 (DOI)000384425400004 ()27383542 (PubMedID)
Funder
Swedish Research CouncilEU, European Research Council, 306474Swedish National Infrastructure for Computing (SNIC), SNIC2015-16-12EU, FP7, Seventh Framework Programme, 283570
Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2017-11-28Bibliographically approved
5. Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
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2018 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
Abstract [en]

The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl­propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme
Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-12-03Bibliographically approved
6. Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.
Open this publication in new window or tab >>Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.
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(English)Manuscript (preprint) (Other academic)
National Category
Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-325490 (URN)
Available from: 2017-07-02 Created: 2017-07-02 Last updated: 2017-07-03

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