ISOLATION AND CHARACTERIZATION OF A CDNA-ENCODING RAT-LIVER CYTOSOLICEPOXIDE HYDROLASE AND ITS FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI

A cDNA of 1992 base pairs encoding the complete rat liver cytosolic ep oxide hydrolase has been isolated using a polymerase chain reaction-de rived DNA fragment (Arand, M., Vnehr, M., Thomas, H., Zeller, H. D., a nd Oesch, F. (1991) FEBS Lett. 294, 19-22) known to represent the 3'-e nd of the cytosolic epoxide hydrolase mRNA. Sequence analysis revealed an open reading frame of 1662 nucleotides corresponding to 554 amino acids (M(r) = 62,268). The DNA sequence obtained did not display signi ficant homology to the sequences of microsomal epoxide hydrolase or le ukotriene A4 hydrolase or to any other DNA included in the EMBL Data B ank (release 32). On Northern blotting of rat liver RNA, a single mRNA species was detected that was strongly induced on treatment of the an imal with fenofibrate, a potent peroxisome proliferator. The most sign ificant structure of the deduced protein is a modified peroxisomal tar geting signal (Ser-Lys-Ile) at the carboxyl terminus that is regarded to be responsible for the unusual dual localization of the cytosolic e poxide hydrolase in peroxisomes as well as in the cytosol. In addition , a leucine zipper-like motif was identified at the amino terminus. It s possible implication for the observed dimeric structure of cytosolic epoxide hydrolase is discussed. The isolated cDNA was expressed in ba cteria to yield a catalytically active enzyme. Specific activity of th e crude lysate obtained exceeded that of rat liver cytosols from maxim ally induced animals by a factor of 8.