The N-terminal domain of mammalian soluble epoxide hydrolase is a phosphatase

Abstract

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.

Insights into the Catalytic Mechanism of Human sEH Phosphatase by Site-Directed Mutagenesis and LC–MS/MS Analysis

“…All experiments were performed in triplicate from three separately produced and purified protein batches. The sEH WT phosphatase hydrolysed 4-NPP with a turnover number of 0.033 x s -1 and a K m of 1.04 mM, these values are of the same order of magnitude as previously found in the sEH phosphatase 12 . Table 1 summarises the kinetic parameters derived from the analysis of the sEH mutant proteins.…”

“…On the basis of structural similarities between the sEH and other members of the HAD superfamily as described in detail earlier 12 , we predicted candidate catalytic amino acids in the active site of the sEH phosphatase domain to be crucial to its catalytic activity. The sEH phosphatase has four conserved sequence motifs as highlighted in figure 2.…”

“…Due to the His 6 -tag (The His 6 -tag was shown previously not to interfere with the catalytic activity of the enzyme 12 ) the sEH proteins were purified in a two step procedure using metal chelate affinity chromatography followed by size exclusion chromatography. As an example, figure 3 gives an…”

Müller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid

“…The apparent difference in the sEH / and sEH iKO retinas can be most likely attributed to the fact that the sEH was still expressed at day 2 in the latter group of animals. The mammalian sEH protein is a homodimer, and each monomer consists of an N-terminal domain which displays lipid phosphatase activity and a larger C terminal which possesses classical /-hydrolase activity (Cronin et al, 2003;Newman et al, 2003). Therefore, in a second approach to ensure that the defects observed in the sEH / mice were due to the loss of epoxide hydrolase activity, newborn wild-type mice were treated with trans-4- [4-(3-adamantan-1-ylureido)cyclohexyloxy]-benzoic acid (t-AUCB), a specific sEH inhibitor which does not affect the activity of the lipid phosphatase domain (Hwang highest sEH activity was detected in the liver, activity in the retina was greater than that detected in either the spleen or lung (Fig.…”

Soluble Epoxide Hydrolase: Potential Target for Inflammation and Inflammation-Driven Cancer

“…EPHX-2 is located on chromosome 8p21-p12, nearly 45kb in length and consists of 19 exons. sEH protein is 555 amino acids and has both an N-terminal phosphatase activity that catalyzes the lipid backbone and a C-terminal [13]. The catalytic mechanism involves formation of a covalent alkylenzyme ester intermediate as a result of nucleophilic attack by Asp333.…”