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The Saccharomyces cerevisiae ORF YNR064c protein has characteristics of an 'orphaned' epoxide hydrolase
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
2005 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1748, 213-221 p.Article in journal (Refereed) Published
Abstract [en]

The open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterialdehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the a/hhydrolasefold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of theprotein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c proteindisplayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide.Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a newsubclass of a/h-hydrolase fold enzymes.

Place, publisher, year, edition, pages
2005. Vol. 1748, 213-221 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-93346DOI: 10.1016/j.bbapap.2005.01.005OAI: oai:DiVA.org:uu-93346DiVA: diva2:166799
Available from: 2005-09-09 Created: 2005-09-09 Last updated: 2017-12-14Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 80
Keyword
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
Identifiers
urn:nbn:se:uu:diva-5900 (URN)91-554-6315-0 (ISBN)
Public defence
2005-09-30, B22, BMC, Husarg. 5, Uppsala, 13:15
Opponent
Supervisors
Available from: 2005-09-09 Created: 2005-09-09Bibliographically approved

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Widersten, Mikael

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