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X-ray structure of potato epoxide hydrolase sheds light on its substrate specificity
Swedish University of Agricultural Sciences, Department of Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2006 (English)In: Protein Science, ISSN 0961-8368, Vol. 15, no 7, 1628-1637 p.Article in journal (Refereed) Published
Abstract [en]

Abstract: Epoxide hydrolases catalyze the conversion of epoxides to diols. The known functions of such enzymes include detoxification of xenobiotics, drug metabolism, synthesis of signaling compounds, and intermediary metabolism. In plants, epoxide hydrolases are thought to participate in general defense systems. In the present study, we report the first structure of a plant epoxide hydrolase, one of the four homologous enzymes found in potato. The structure was solved by molecular replacement and refined to a resolution of 1.95 angstrom. Analysis of the structure allows a better understanding of the observed substrate specificities and activity. Further, comparisons with mammalian and fungal epoxide hydrolase structures reported earlier show the basis of differing substrate specificities in the various epoxide hydrolase subfamilies. Most plant enzymes, like the potato epoxide hydrolase, are expected to be monomers with a preference for substrates with long lipid-like substituents of the epoxide ring. The significance of these results in the context of biological roles and industrial applications is discussed.

Place, publisher, year, edition, pages
2006. Vol. 15, no 7, 1628-1637 p.
Keyword [en]
X-ray crystallography, epoxide hydrolase, active site, trans-stilbene oxide, substrate specificity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
URN: urn:nbn:se:uu:diva-93349DOI: 10.1110/ps.051792106ISI: 000238707200006OAI: oai:DiVA.org:uu-93349DiVA: diva2:166802
Available from: 2005-09-09 Created: 2005-09-09 Last updated: 2016-05-09Bibliographically approved
In thesis
1. Characterization of Epoxide Hydrolases from Yeast and Potato
Open this publication in new window or tab >>Characterization of Epoxide Hydrolases from Yeast and Potato
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Epoxides are three-membered cyclic ethers formed in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to yield the corresponding diols. EHs have been implicated in diverse functions such as detoxification of various toxic epoxides, as well as regulation of signal substance levels.

The main goal of this thesis was to investigate and characterize the α/β hydrolase fold EH. The first part concerns the identifictaion of an EH in Saccharomyces cerevisiae. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1.

Despite the important function of EH, no EH has previously been established in S. cerevisiae. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The S. cerevisiae protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates.

In plants, EHs are involved in the general defence system, both in the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly expressed wild type and mutant potato EH were investigated in detail using the two enantiomers of trans-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the S,S-enantiomer of trans-stilbene oxide. Furthermore, distinct pH dependence of the two enantiomers probably reflects differences in the microscopic rate constants of the substrates. The detailed function of the two catalytic tyrosines was also studied. The behavior of the tyrosine pair resembles that of a bidentate Lewis acid and we conclude that these tyrosines function as Lewis acids rather then proton donors.

The three dimensional structure of StEH1 was solved, representing the first structure of a plant EH. The structure provided information about the substrate specificity of StEH1.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 50 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 80
Biochemistry, Epoxide hydrolase, Catalytic residues, Active site, trans-stilbene oxide, Rapid kinetics, Active site tyrosyls, Enzyme mechanism, Lewis acid, X-ray crystallography, Substrate specificity, Unidentified ORF, α/β hydrolase fold, Saccharomyces cerevisiae, Biokemi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-5900 (URN)91-554-6315-0 (ISBN)
Public defence
2005-09-30, B22, BMC, Husarg. 5, Uppsala, 13:15
Available from: 2005-09-09 Created: 2005-09-09Bibliographically approved

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Mowbray, Sherry L.Widersten, Mikael
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