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Published online before print May 30, 2008, 10.1110/ps.034173.107
Protein Science (2008), 17:1275-1284. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 The Protein Society
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Removal of distal protein–water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability

Ann Thomaeus1, Agata Naworyta2, Sherry L. Mowbray2, and Mikael Widersten1

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


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