Expanding the catalytic triad in epoxide hydrolases and related enzymes
2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, 5702-5713 p.Article in journal (Refereed) Published
Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.
Place, publisher, year, edition, pages
2015. Vol. 5, no 10, 5702-5713 p.
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-260232DOI: 10.1021/acscatal.5b01639ISI: 000362391500006OAI: oai:DiVA.org:uu-260232DiVA: diva2:846735
FunderEU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12