Epoxides are three-membered cyclic ethers created in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes which catalyze the hydrolysis of epoxides to yield the related diols. EHs have been implicated in diverse functions like detoxification of different toxic epoxides, in addition to regulation of signal substance levels.The primary objective of this dissertation was to look into and characterize the a/ß hydrolase fold EH. Part one concerns the identifictaion of an EH in Saccharomyces cerevisiae. The subsequent part consists of comprehensive mechanistic and structural studies of a plant EH from potato, StEH1.Regardless of the significant function of EH, no EH has formerly been established in S. cerevisiae. By sequence analysis, we now have determined a new subclass of EH found in yeast and in a number of microorganisms. The S. cerevisiae protein was generated recombinantly and was shown to present low catalytic activity with tested epoxide substrates.In plants, EHs are associated with the general defence system, in both the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly indicated wild type and mutant potato EH were researched at length using the 2 enantiomers of trans-stilbene oxide (TSO). The suggested catalytic residues of StEH1 were verified. StEH1 is slightly enantioselective for the S,S-enantiomer of trans-stilbene oxide. In addition, distinct pH dependence of the 2 enantiomers most likely echos variations in the microscopic rate constants of the substrates. The comprehensive function of the 2 catalytic tyrosines was also analyzed. The behaviour of the tyrosine pair resembles that of a bidentate Lewis acid and we deduce that these tyrosines work as Lewis acids instead of proton donors.The 3 dimensional structure of StEH1 was solved, symbolizing the first structure of a plant EH. The structure offered details about the substrate specificity of StEH1.
Contents
Introduction
Enzymes
Epoxides
Epoxide hydrolases
Mammalian mEH and sEH
Plant epoxide hydrolases
Microbial EH
Catalytic mechanism of epoxide hydrolases
Three-dimensional structure
Present investigation
Aims of the study
Identification of an epoxide hydrolase in S. cerevisiae (Paper I
Introduction to the yeast project
EH in yeast
Endogenous and recombinant expression of YNR064c
Characterization of YNR064c
Conclusions regarding YNR064c
Characterization of the catalytic mechanism of potato EH, StEH1 (Paper II, III and IV)
Introduction to the potato project
Expression and purification of StEH1 and mutants (Paper II)
Catalytic residues of StEH1
Catalytic mechanism of StEH1
Hydrolysis of 9,10-epoxystearate
The catalytic base
pH dependence of kinetic constants for StEH1
Ionization constant of active site tyrosines (Paper III)
The catalytic tyrosines
pH dependence of microscopic rate constants
The X-ray structure of StEH1 (Paper IV)
Overall structure
Active site architecture
Ongoing structural studies…….
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