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Removal of distal protein–water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability

¹ Department of Biochemistry and Organic Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden
² Department of Molecular Biology, Swedish University of Agricultural Sciences, SE-751 24 Uppsala, Sweden

(RECEIVED December 19, 2007; FINAL REVISION April 8, 2008; ACCEPTED April 21, 2008)

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

Abbreviations: StEH1, epoxide hydrolase 1 from Solanum tuberosum; TSO, trans-stilbene oxide; PEG, polyethylene glycol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.034173.107.

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