FURTHER CHARACTERIZATION OF THE PEROXISOMAL 3-HYDROXYACYL-COA DEHYDROGENASES FROM RAT-LIVER - RELATIONSHIP BETWEEN THE DIFFERENT DEHYDROGENASES AND EVIDENCE THAT FATTY-ACIDS AND THE C-27 BILE-ACIDS DI-HYDROXYCOPROSTANIC AND TRI-HYDROXYCOPROSTANIC ACIDS ARE METABOLIZED BY SEPARATE MULTIFUNCTIONAL PROTEINS

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isola ted rat liver peroxisomal fractions. The enzymes were designated I-V a ccording to their order of elution from the first column used in the p urification procedure, Determination of the substrate (L- or D-hydroxy acyl-CoA) stereospecificity and (de)hydratase measurements with the di fferent 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblott ing analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme III revealed the following picture. Rat liver peroxisomes c ontain two multifunctional beta-oxidation proteins: (a) multifunctiona l protein 1 (the classical multifunctional protein; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and Delta(3), Delta(2)-enoyl-CoA isomerase activity (enzyme IV) and (b) multifuncti onal protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydrox yacyl-CoA dehydrogenase activity (enzyme III). Because of their substr ate stereospecificity and because of the stereochemical configuration of the naturally occurring beta-oxidation intermediates, MFP-1 and MFP -2 appear to be involved in the beta-oxidation of fatty acids and bile acids intermediates, respectively. The deduced amino acid sequence of the cloned MFP-2 cDNA is highly similar to that of the recently descr ibed porcine endometrial estradiol 17 beta-dehydrogenase [Leenders, F. , Adamski, J., Husen, B., Thole, H. H. & Jungblut, P. W. (1994) Eur. J . Biochem. 222, 221-227], In agreement, MFP-2 also displayed estradiol 17 beta-dehydrogenase activity, indicating that MFP-2, and the steroi d dehydrogenase are identical enzymes. MFP-2 is partially cleaved, mos t probably in vivo, in a estradiol 17 beta-dehydrogenase/D-3-hydroxyac yl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hyd ratase. The physiological significance of enzyme I in bile acid synthe sis (and steroid metabolism) remains to be determined. MFP-1 (enzyme I V) is artefactually cleaved during purification giving rise to 3-hydro xyacyl-CoA dehydrogenase V. Hydroxyacyl-CoA dehydrogenase II is a mito chondrial contaminant similar to porcine and murine mitochondrial 3-hy droxyacyl-CoA dehydrogenase.