Molecular Cloning and Expression of a 2-Arylpropionyl-Coenzyme A Epimerase: A Key Enzyme in the Inversion Metabolism of Ibuprofen
- Department of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen/Nürnberg, D-91054 Erlangen, Germany
Abstract
The 2-arylpropionic acid derivatives, including ibuprofen, are the most widely used anti-inflammatory analgesic cyclooxygenase inhibitors. The (−)-R-enantiomer, which is inactive in terms of cyclooxygenase inhibition, is epimerized in vivo via the 2-arylpropionyl-coenzyme A (CoA) epimerase to the cyclooxygenase-inhibiting (+)-S-enantiomer. The molecular biology of the epimerization pathway is largely unknown. To clarify this mechanism, the sequence of the 2-arylpropionyl-CoA epimerase was identified, and the enzyme cloned and expressed. A cDNA clone encoding the 2-arylpropionyl-CoA epimerase was isolated from a rat liver cDNA library. The nucleotide and the deduced amino acid sequence of this enzyme was determined. Significant amino acid sequence similarity was found between the rat epimerase and carnitine dehydratases from Caenorhabditis elegans (41%) andEscherichia coli (27%). A bacterial expression system (E. coli strain M15[pREP4]) was used to express the epimerase protein, representing up to 20–30% of the total cellularE. coli protein. The expression of the epimerase was confirmed with Western blots 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 addition to its obvious importance in drug metabolism, the homology of the epimerase with carnitine dehydratases from several species suggests that this protein, which up to now has only been characterized as having a role in drug transformation, has a function in lipid metabolism.
Footnotes
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Send reprint requests to: Gerd Geisslinger, Ph.D., M. D., Department of Experimental and Clinical Pharmacology and Toxicology, Universitätsstr. 22, D-91054 Erlangen, Germany. E-mail:gg{at}macpost.pharmakologie.uni-erlangen.de
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 353/A9) and by Graduiertenkolleg/Schmerzforschung.
- Abbreviations:
- IBU
- ibuprofen
- PCR
- polymerase chain reaction
- (R)-IBU-CoA
- (R)-ibuprofenoyl-coenzyme A thioester
- DIG
- digoxigenin
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
- COX
- cyclooxygenase
- IPTG
- isopropylthiogalactoside
- TBS-T
- Tris-buffered saline/Tween 20
- bp
- base pair(s)
- kb
- kilobase pair(s)
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- Received September 3, 1996.
- Accepted December 13, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
