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Doctoral thesis, 2010

Structure-function studies of epoxide hydrolases

Naworyta, Agata

Abstract

Epoxides are three-membered cyclic ethers formed in cells via several metabolic pathways. Epoxide hydrolases (EHs) are enzymes that hydrolyse epoxides to the corresponding diols. The main goal of this thesis was to investigate the structures of EHs from the alpha/beta-hydrolase family. The first part concerns the structural and functional analysis of a protein-water channel found in EHs in many plants. Thermostability studies, sequence analysis and determination of the x-ray structure of a mutated EH enzyme from Solanum tuberosum led to the conclusions that the water channel in plants participates in stabilization of the protein structure and furthermore, it forms an efficient system to enable transfer of protons that are required for enzymatic catalysis. The second part describes how computational methods together with structural and kinetic information identified factors that are responsible for the enhanced enantioselectivity of an improved variant of EH from Aspergillus niger obtained during a directed evolution process. The x-ray structure of the mutant showed that dramatic changes in the active site explain why the preferred (S)-substrate binds more easily in the active site than the disfavored (R)-enantiomer. The study underscores the importance of obtaining structural data when attempting to understand the results of directed evolution. The last part presents the structures of two novel microbial EHs that have been shown to produce chemically valuable 1,2-diols and exhibit high enantioselectivity. Their similarity to the mammalian microsomal EH, a key enzyme in detoxification, provided new information about its possible structure. The improved sequence alignment based on the structural work gives new insights on the connections between sequences/structures and the broad scope of selectivities among EHs

Keywords

epoxy compounds; hydrolases; enzymatic hydrolysis; chemical structure; crystallization; computer applications; solanum tuberosum

Published in

Acta Universitatis Agriculturae Sueciae
2010, number: 2010:5
ISBN: 978-91-576-7482-1
Publisher: Department of Molecular Biology, Swedish University of Agricultural Sciences

Authors' information

Naworyta, Agata
Swedish University of Agricultural Sciences, Department of Molecular Biology

UKÄ Subject classification

Biochemistry and Molecular Biology

URI (permanent link to this page)

https://res.slu.se/id/publ/28608