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Active site of epoxide hydrolases revisited: A noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 9, 2466-2479 p.Article in journal (Refereed) Published
Abstract [en]

The carboxylate of Glu(35) in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of k(cat), and the rate of alkylenzyme hydrolysis, k(3). The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pK(a) of His(300) in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu(35) participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His(300).

Place, publisher, year, edition, pages
2007. Vol. 46, no 9, 2466-2479 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-97211DOI: 10.1021/bi062052sISI: 000244468000020PubMedID: 17284015OAI: oai:DiVA.org:uu-97211DiVA: diva2:172045
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Challenges in Computational Biochemistry: Solvation and Ligand Binding
Open this publication in new window or tab >>Challenges in Computational Biochemistry: Solvation and Ligand Binding
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.

The thermodynamic integration technique is used to calculate pKa values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated pKa values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine pKa shifts in wild-type and mutant epoxide hydrolase. The calculated pKa values support a model that can explain some of the pH dependent properties of this enzyme.

Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 432
Keyword
Molecular biology, computer simulations, molecular dynamics, solvation free energy, Generalized-Born, Poisson-Boltzmann, ligand binding, binding free energy, linear interaction energy, binding entropy, hydration entropy, Molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-8738 (URN)978-91-554-7200-9 (ISBN)
Public defence
2008-05-23, B7:101, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-04-29 Created: 2008-04-29Bibliographically approved
2. Biochemical Studies on a Plant Epoxide Hydrolase: Discovery of a Proton Entry and Exit Pathway and the Use of In vitro Evolution to Shift Enantioselectivity
Open this publication in new window or tab >>Biochemical Studies on a Plant Epoxide Hydrolase: Discovery of a Proton Entry and Exit Pathway and the Use of In vitro Evolution to Shift Enantioselectivity
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase.

Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis.

In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base.

In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein.

Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 739
Keyword
epoxide hydrolase, enantioselectivity, in vitro evolution, proton wire, epoxides, selectivity, CASTing, structure-function
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-122424 (URN)978-91-554-7796-7 (ISBN)
Public defence
2010-05-21, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2010-04-29 Created: 2010-04-12 Last updated: 2010-04-29Bibliographically approved

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