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

Abstract

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.

An insect farnesyl phosphatase homologous to the N-terminal domain of soluble epoxide hydrolase

“…Alignment of these (Fig. 1A) showed that they had one or more conserved motifs typical of phosphatases including; 1) an aspartic acid residue as the catalytic nucleophile, 2) a serine or threonine for binding the phosphate group, and, 3) two aspartic acid residues thought to be important for Mg 2+ binding [7, 11]. Two phosphatases (Phos17294 and Phos10352) were eliminated from further analysis because they lacked the second serine or threonine conserved motif.…”

The Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase

“…To demonstrate covalent catalysis in the H18D variant, we first sought to obtain direct evidence for the formation of a phospho-enzyme intermediate. Using a 32 P-labeled phosphate group 26,49 in the Glc1P substrate, we could clearly show the incorporation of radiolabel into the wildtype enzyme, but did not observe the same for the H18D variant (Figure S2). The H18A variant did not incorporate radiolabel, as expected from the requirement of an enzyme nucleophile to form a phosphorylated enzyme.…”

“…49,50 We therefore applied chemical reduction with sodium borohydride to convert aspartylphosphate into the non-labile homoserine (Figure S3a) and analyzed the enzyme by tryptic peptide mass fingerprinting. 49 For HAD13 incubated with Glc1P, indeed, the expected peptide containing the catalytic nucleophile Asp9 present as homoserine was found in high abundance (Figure S3 and Table S3). The same peptide was not detectable for the control (HAD13 incubated in absence of Glc1P, Table S4).…”

Bacterial Expression and HTS Assessment of Soluble Epoxide Hydrolase Phosphatase

“…Furthermore, it is proposed that the hydrolase domain of sEH regulates the activity of sEH-P 30 and vice versa. Thus, it was important to develop an assay system with an isolated N-terminal sEH-P domain to exclude the influence of the C-terminal domain.…”

“…3,4 The sEH-P has structural and sequential similarity to the haloacid dehalogenase family and cleaves phosphate esters in a magnesium-dependent nucleophilic attack by Asp9. 5 Endogenic substrates of sEH-P are isoprenoid phosphates 6 and lysophosphatidic acids. 7,8 Phenotypic differences between mice treated with sEH-H inhibitors and sEH knockout mice, which lack both domains, indicate a role of sEH-P in hypercholesterolemia 9 and pulmonary hypertension.…”