The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in which the CYP2B1 membrane anchor signal sequence replaced the N-terminal 20 amino acid residues of mEH, was co-translationally inserted into dog pancreas microsomes in a cell-free translation system, whereas a truncated epoxide hydrolase with a deletion of the 20 N-terminal amino acid residues was not co-translationally inserted. (ii) The mEH and the CYP2B1-mEH fusion protein, but not the truncated epoxide hydrolase, were anchored in microsomes in a cell-free translation system and in membrane fractions derived from fibroblasts which expressed these proteins heterologously. These fibroblasts were also used to evaluate the significance of the mEH membrane anchor for the catalytic activity of mEH. The mEH, the truncated mEH and the CYP-EH fusion protein were found to be enzymically active. This result shows that the membrane anchor signal sequence of mEH is dispensable for the catalytic activity of this protein. However, truncated mEH was only expressed at low levels, which might indicate that this protein is unstable.

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Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.

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