title: Bacterial Expression and HTS Assessment of Soluble Epoxide Hydrolase Phosphatase.
abstract: Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were identified.
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany.
authors.forename: Franca-Maria
authors.initials: FM
authors.lastname: Klingler
authors.affiliation: Fraunhofer Institute for Molecular Biology and Environmental Ecology ScreeningPort, Hamburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Wolf
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Wittmann
authors.affiliation: Fraunhofer Institute for Molecular Biology and Environmental Ecology ScreeningPort, Hamburg, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Gribbon
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany proschak@pharmchem.uni-frankfurt.de.
authors.forename: Ewgenij
authors.initials: E
authors.lastname: Proschak
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics; D012995:Solubility
chemical_list: D004791:Enzyme Inhibitors; D010744:Phosphoric Monoester Hydrolases; D004851:Epoxide Hydrolases; C506428:EPHX2 protein, human
keywords: enzyme assay; high-throughput screening; phosphatase; soluble epoxide hydrolase