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Expanding the catalytic triad in epoxide hydrolases and related enzymes
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. (Kamerlin)
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. (Kamerlin)
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. (Kamerlin)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Widersten)
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2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, 5702-5713 p.Article 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.

Place, publisher, year, edition, pages
2015. Vol. 5, no 10, 5702-5713 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-260232DOI: 10.1021/acscatal.5b01639ISI: 000362391500006OAI: oai:DiVA.org:uu-260232DiVA: diva2:846735
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
In thesis
1. Towards Understanding of Selectivity & Enantioconvergence of an Epoxide Hydrolase
Open this publication in new window or tab >>Towards Understanding of Selectivity & Enantioconvergence of an Epoxide Hydrolase
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1).

A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.   

The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104.

Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering.

For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 90 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1378
Keyword
Epoxide hydrolase, Epoxide, Enantioselectivity, Regioselectivity, Enantioconvergence, Crystal structures, Biocatalysis, Immobilization, Transient kinetics, CASTing, Directed evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-286557 (URN)978-91-554-9586-2 (ISBN)
Public defence
2016-06-10, A1:107a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2016-05-20 Created: 2016-04-21 Last updated: 2016-06-15
2. Extending the Reach of Computational Approaches to Model Enzyme Catalysis
Open this publication in new window or tab >>Extending the Reach of Computational Approaches to Model Enzyme Catalysis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically:

  1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems.

  2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1.

  3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example.

In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1484
Keyword
epoxide hydrolase, enantioselectivity, regioselectivity, enantioconvergence, biocatalysis, empirical valence bond, computational directed evolution
National Category
Theoretical Chemistry Biochemistry and Molecular Biology Structural Biology
Identifiers
urn:nbn:se:uu:diva-314686 (URN)978-91-554-9816-0 (ISBN)
Public defence
2017-03-24, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
EU, European Research Council, 306474
Available from: 2017-03-02 Created: 2017-02-04 Last updated: 2017-03-06
3. Computational modelling of enzyme selectivity
Open this publication in new window or tab >>Computational modelling of enzyme selectivity
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. 104 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1530
Keyword
enantiomer, epoxide hydrolase, chiral catalysis, empirical valence bond approach, method development
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-326108 (URN)978-91-513-0005-4 (ISBN)
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: 2017-09-15

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Amrein, Beat A.Bauer, PaulDuarte, FernandaJanfalk Carlsson, ÅsaMowbray, Sherry L.Widersten, MikaelKamerlin, Shina C. L.

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