Further Characterization of the Peroxisomal 3‐Hydroxyacyl‐Coa Dehydrogenases from Rat Liver

Abstract

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I-V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (L-or o-hydroxyacyl-CoA) stereospecificity and (de)hydratase measurements with the different 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme I11 revealed the following picture.Rat liver peroxisomes contain two multifunctional P-oxidation proteins : (a) multifunctional protein 1 (the classical multifunctional protein ; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and A', A'-enoyl-CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activity (enzyme LII).Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring P-oxidation intermediates, MFP-1 and MFP-2 appear to be involved in the P-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 described porcine endometrial estradiol 17P-dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, MFP-2 is partially cleaved, most probably in vivo, in a estradiol 17P-dehydrogenasel~-3-hydroxyacyl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP-1 (enzyme IV) is artefactually cleaved during purification giving rise to 3-hydroxyacyl-CoA dehydrogenase V. 3-Hydroxyacyl-CoA dehydrogenase I1 is a mitochondrial contaminant similar to porcine and murine mitochondrial 3-hydroxyacyl-CoA dehydrogenase.

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

“…Samples were further prepared according to the instructions for electrophoresis of NuPAGE Bis-Tris mini gels Hsd17b4 seems to be able to handle all peroxisomal substrates ( 7 ), which is in line with earlier biochemical studies using purifi ed enzymes (8)(9)(10)(11)(12). Indeed, HSD17B4-deficient patients and Hsd17b4 KO mice accumulate very long-chain as well as branched-chain fatty acids and bile acid intermediates ( 13,14 ).…”

Participation of Two Members of the Very Long-chain Acyl-CoA Synthetase Family in Bile Acid Synthesis and Recycling

“…In rodents, distinct enzymes carry out these two processes (29). D-bifunctional protein catalyzes the second (enoyl-CoA hydratase) and third (hydroxyacyl-CoA dehydrogenase) activities (30). The thiolase associated with the 58-kDa protein sterol carrier protein X is thought to carry out the final reaction, thiolytic cleavage and release of propionyl-CoA (31).…”

Development of a New Enzymatic Diagnosis Method for Very-long-chain Acyl-CoA Dehydrogenase Deficiency by Detecting 2-Hexadecenoyl-CoA Production and its Application in Tandem Mass Spectrometry-based Selective Screening and Newborn Screening in Japan

“…We synthesized 2-hexadecenoyl-CoA from palmitoyl-CoA using ACO in a modified version of the method described by Dieuaide-Noubhani et al (11). The reaction mixture, which contained 50 mM K 2 HPO 4 buffer (pH 7.4), 0.1 mg ACO, and 0.2 mM palmitoyl-CoA, was incubated at 37°C for 15 min.…”