MOLECULAR-CLONING AND EXPRESSION OF A 2-ARYLPROPIONYL-COENZYME-A EPIMERASE - A KEY ENZYME IN THE INVERSION METABOLISM OF IBUPROFEN

The 2-arylpropionic acid derivatives, including ibuprofen, are the mos t widely used anti-inflammatory analgesic cyclooxygenase inhibitors. T he (-)-R-enantiomer, which is inactive in terms of cyclooxygenase inhi bition, is epimerized in vivo via the 2-arylpropionyl-coenzyme A (CoA) epimerase to the cyclooxygenase-inhibiting (+)-S-enantiomer. The mole cular biology of the epimerization pathway is largely unknown. To clar ify this mechanism, the sequence of the 2-arylpropionyl-CoA epimerase was identified, and the enzyme cloned and expressed. A cDNA clone enco ding the 2-aryipropionyl-CoA epimerase was isolated from a rat liver c DNA library. The nucleotide and the deduced amino acid sequence of thi s enzyme was determined. Significant amino acid sequence similarity wa s found between the rat epimerase and carnitine dehydratases from Caen orhabditis elegans (41%) and Escherichia coli (27%). A bacterial expre ssion system (E. coli strain M15[pREP4]) was used to express the epime rase protein, representing up to 20-30% of the total cellular E. coil protein. The expression of the epimerase was confirmed with Western bl ots using specific anti-epimerase antibodies and by measuring the rate of inversion of (R)-ibuprofenoyl-CoA. Northern blot analysis revealed a prominent 1.9-kb mRNA transcript in different rat tissues. In addit ion to its obvious importance in drug metabolism the homology of the e pimerase with carnitine dehydratases from several species suggests tha t this protein, which up to now has only been characterized as having a role in drug transformation, has a function in lipid metabolism.