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Catalysis of potato epoxide hydrolase, StEH1
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: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 390, 633-640 p.Article in journal (Refereed) Published
Abstract [en]

The kinetic mechanism of epoxide hydrolase (EC from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 mmol·min-1·mg-1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

Place, publisher, year, edition, pages
2005. Vol. 390, 633-640 p.
Keyword [en]
active site, catalysis, epoxide hydrolase, rapid
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-93347DOI: 10.1042/BJ20050526PubMedID: 15882148OAI: oai:DiVA.org:uu-93347DiVA: diva2:166800
Available from: 2005-09-09 Created: 2005-09-09 Last updated: 2010-06-18Bibliographically 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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