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Modification of substrate specificity resulted in an epoxide hydrolase with shifted enantiopreference for (2,3-epoxypropyl)benzene
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2010 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 10, 1422-1429 p.Article in journal (Refereed) Published
Abstract [en]

Random mutagenesis targeted at hot spots of non-catalytic active-site residues of potato epoxide hydrolase StEH1 combined with an enzyme-activity screen allowed for isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the variant displaying most radical functional changes, showed a 15:1 preference for the R-enantiomer. This mutant had accumulated four substitutions distributed to two, out of four mutated, hot spots: W106L, L109Y, V141K and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S-enantiomer combined with retained activity with the R-enantiomer. The results demonstrate the feasibility to mold stereoselectivity in this biocatalytically relevant enzyme.

Place, publisher, year, edition, pages
2010. Vol. 11, no 10, 1422-1429 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-122306DOI: 10.1002/cbic.201000185ISI: 000280787400017OAI: oai:DiVA.org:uu-122306DiVA: diva2:309635
Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-12-12Bibliographically approved
In thesis
1. 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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Gurell, AnnWidersten, Mikael

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