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Biochemical Studies on a Plant Epoxide Hydrolase: Discovery of a Proton Entry and Exit Pathway and the Use of In vitro Evolution to Shift Enantioselectivity
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Widersten)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase.

Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis.

In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base.

In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein.

Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 739
Keywords [en]
epoxide hydrolase, enantioselectivity, in vitro evolution, proton wire, epoxides, selectivity, CASTing, structure-function
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-122424ISBN: 978-91-554-7796-7 (print)OAI: oai:DiVA.org:uu-122424DiVA, id: diva2:310082
Public defence
2010-05-21, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2010-04-29 Created: 2010-04-12 Last updated: 2010-04-29Bibliographically approved
List of papers
1. Active site of epoxide hydrolases revisited: A noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate
Open this publication in new window or tab >>Active site of epoxide hydrolases revisited: A noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 9, p. 2466-2479Article in journal (Refereed) Published
Abstract [en]

The carboxylate of Glu(35) in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of k(cat), and the rate of alkylenzyme hydrolysis, k(3). The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pK(a) of His(300) in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu(35) participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His(300).

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-97211 (URN)10.1021/bi062052s (DOI)000244468000020 ()17284015 (PubMedID)
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
2. Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability
Open this publication in new window or tab >>Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability
2008 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 17, no 7, p. 1275-1284Article in journal (Refereed) Published
Abstract [en]

A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro186, Leu266, His269, and the His153 imidazole. The hydroxyl of Tyr149 is also an integrated component of the chain, which leads to the hydroxyl of Tyr154. Available data suggest that Tyr154 functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 Å resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k cat/K M was similar or slightly increased compared with the wild-type reactions. k cat for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K M. Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k cat but lowered thermostability.

Keywords
epoxide hydrolase, proton wire, thermostability, mutants, X-ray crystal structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-17590 (URN)10.1110/ps.034173.107 (DOI)000257110700017 ()18515642 (PubMedID)
Available from: 2008-07-11 Created: 2008-07-11 Last updated: 2017-12-08Bibliographically approved
3. Modification of substrate specificity resulted in an epoxide hydrolase with shifted enantiopreference for (2,3-epoxypropyl)benzene
Open this publication in new window or tab >>Modification of substrate specificity resulted in an epoxide hydrolase with shifted enantiopreference for (2,3-epoxypropyl)benzene
2010 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 10, p. 1422-1429Article in journal (Refereed) Published
Abstract [en]

Random mutagenesis targeted at hot spots of non-catalytic active-site residues of potato epoxide hydrolase StEH1 combined with an enzyme-activity screen allowed for isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the variant displaying most radical functional changes, showed a 15:1 preference for the R-enantiomer. This mutant had accumulated four substitutions distributed to two, out of four mutated, hot spots: W106L, L109Y, V141K and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S-enantiomer combined with retained activity with the R-enantiomer. The results demonstrate the feasibility to mold stereoselectivity in this biocatalytically relevant enzyme.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-122306 (URN)10.1002/cbic.201000185 (DOI)000280787400017 ()
Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-12-12Bibliographically approved

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