The mammalian soluble epoxide hydrolase (sEH) is an enzyme with multiple functions, being implicated in detoxification of xenobiotic epoxides as well as in regulation of physiological processes such as blood pressure. The enzyme is a homodimer, in which each subunit is composed of two domains. The 35-kDa C-terminal domain has an ␣͞␤ hydrolase fold and harbors the catalytic center for the EH activity. The 25-kDa N-terminal domain has a different ␣͞␤ fold and belongs to the haloacid dehalogenase superfamily of enzymes. The catalytic properties of the enzyme reported so far can all be explained by the action of the C-terminal domain alone. The function of the N-terminal domain, other than in structural stabilization of the dimer, has therefore remained unclear. By structural comparison of this domain to other haloacid dehalogenase family members, we identified a putative active site containing all necessary components for phosphatase activity. Subsequently, we found rat sEH hydrolyzed 4-nitrophenyl phosphate with a rate constant of 0.8 s ؊1 and a Km of 0.24 mM. Recombinant human sEH lacking the C-terminal domain also displayed phosphatase activity. Presence of a phosphatase substrate did not affect epoxide turnover nor did epoxides affect dephosphorylation by the intact enzyme, indicating both catalytic sites act independently. The enzyme was unable to hydrolyze 4-nitrophenyl sulfate, suggesting its role in xenobiotic metabolism does not extend beyond phosphates. Thus, we propose this domain participates instead in the regulation of the physiological functions associated with sEH.

The bifunctional human soluble epoxide hydrolase (sEH) is implicated as a regulator of diverse physiological processes due to the brakedown of arachidonic acid derived signalling molecules like epoxyeicosatrienoic acids (EETs) by its epoxide hydrolase domain. Recently, we discovered that the sEH N‐terminal domain displays a novel phosphatase activity. sEH accepts the generic substrate 4‐NPP as well as lipid phosphates, but the physiological role remains uncertain. The phosphatase domain contains three conserved sequence motifs, including the potential catalytic nucleophile Asp9, and several residues implicated in substrate turnover and/or Mg2+ binding. To enlighten the proposed catalytic mechanism we constructed active site mutants by site directed mutagenesis, which were recombinantly expressed as soluble proteins, purified and analysed for their kinetic properties. An exchange of Asp9, Lys160, Asp184 or Asn189 results in a complete loss of phosphatase activity, emphasising the requirement of these amino acids for catalysis, whereas a substitution of Asp11, Thr123, Asn124 and Asp185 leads to sEH mutant proteins with residual phosphatase activity. To confirm the role of Asp9 as catalytic nucleophile we presently analyze the presumed phosphoester intermediate by mass spectrometry. The dual phosphatase and epoxide hydrolase activities give new insights into the physiological role of human sEH.

The bifunctional human soluble epoxide hydrolase (sEH) is implicated as a regulator of diverse physiological processes due to the brakedown of arachidonic acid derived signalling molecules like epoxyeicosatrienoic acids (EETs) by its epoxide hydrolase domain. Recently, we discovered that the sEH N‐terminal domain displays a novel phosphatase activity. sEH accepts the generic substrate 4‐NPP as well as lipid phosphates, but the physiological role remains uncertain.The phosphatase domain contains three conserved sequence motifs, including the potential catalytic nucleophile Asp9, and several residues implicated in substrate turnover and/or Mg2+ binding. To enlighten the proposed catalytic mechanism we constructed active site mutants by site directed mutagenesis, which were recombinantly expressed as soluble proteins, purified and analysed for their kinetic properties. An exchange of Asp9, Lys160, Asp184 or Asn189 results in a complete loss of phosphatase activity, emphasising the requirement of these amino acids for catalysis, whereas a substitution of Asp11, Thr123, Asn124 and Asp185 leads to sEH mutant proteins with residual phosphatase activity. To confirm the role of Asp9 as catalytic nucleophile we presently analyze the presumed phosphoester intermediate by mass spectrometry. The dual phosphatase and epoxide hydrolase activities give new insights into the physiological role of human sEH.