<p>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.</p><p>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 <i>Saccharomyces cerevisiae</i>. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1. </p><p>Despite the important function of EH, no EH has previously been established in <i>S. cerevisiae</i>. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The <i>S. cerevisiae</i> protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates. </p><p>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 <i>trans</i>-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the <i>S,S</i>-enantiomer of<i> trans</i>-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.</p><p>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.</p>

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.

This thesis presents specification and implementation of a system for detection of enzymes in metagenomic data. The detection is based on a provided enzyme sequence and its goal is to search the metagenomic sample for its novel variants. In order to guarantee that found enzymes truly have the desired catalytic function, the system employs a number of catalytic function verification methods. Their specification, implementation and evaluation is one of the main contributions of this thesis. Experiments have shown, that proposed methods reach sensitivity as high as 89%, specificity of 95%, values of AUC metric above 0.9 and average throughput of 1,203 verifications per second on regular personal computer. Evaluation of the system also led to discovery of a partial sequence of novel haloalkane dehalogenase enzyme in a metagenomic sample from soil. The implementation is able to work on a personal computer as well as on a grid computing environment.