Catalysis of potato epoxide hydrolase, StEH1

Abstract

The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 µmol · min −1 · mg −1 with the R,R-or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower K m value. The pH dependences of k cat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis-and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp 105 , Tyr 154 , Tyr 235 and His 300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His 300 → Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

Obtaining Optical Purity for Product Diols in Enzyme-Catalyzed Epoxide Hydrolysis: Contributions from Changes in both Enantio- and Regioselectivity

“…15−18 The latter mechanism is present in the α/β-hydrolase-type enzymes, 19−22 with the ring opening being facilitated by enzyme-provided acids, a Tyr pair. 23,24 Our group is using the potato epoxide hydrolase StEH1 25,26 as the starting scaffold for engineering new epoxide hydrolase enzymes that can be applied as biocatalysts for chiral epoxide ring-opening reactions. 27 StEH1 differs in active-site structure and substrate selectivity from a previously engineered Aspergillus niger epoxide hydrolase.…”

Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis

“…DES solutions were prepared by mixing 0.1 M sodium phosphate with DES after which pH was adjusted to 7.5. StEH1 was purified as described previously (Elfström and Widersten, 2005). Epoxides were dissolved in acetonitrile and added to the reactions mixtures.…”

“…1). To assess effects on enzyme stability, kinetic properties and regioselectivity, the two trans-2-methylstyrene oxide (2-MeSO) enantiomers were tested as substrates for the potato EH StEH1 (Elfström and Widersten, 2005) in the presence of DES cosolvents. The (1S,2S)-enantiomer is a good substrate for StEH1 and was chosen for the activity and stability assays.…”

Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

“…A water molecule, activated by Cif’s histidine-acid pair (His297 and Glu153), then hydrolyzes the ester intermediate to form the corresponding vicinal diol (Bahl et al, 2015a, 2016). For other well-studied EHs, product release is the rate-limiting step, leading to accumulation of the covalently modified enzyme (Elfström and Widersten, 2005; Morisseau and Hammock, 2005). Although only trace quantities of corresponding adduct are observed for WT Cif, the mutant Cif E153Q was expected to permit substrate binding and initial nucleophilic attack, but not hydrolysis.…”