In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an Mr of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578–21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted Mr of 79331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17β-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal β-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase–hydratase–dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of β-oxidation but through reciprocal stereochemical courses.
Peroxisomal multifunctional enzyme of β-oxidation metabolizing d-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization
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Yong-Mei QIN, Matti H. POUTANEN, Heli M. HELANDER, Ari-Pekka KVIST, Kirsi M. SIIVARI, Werner SCHMITZ, Ernst CONZELMANN, Ulf HELLMAN, J. Kalervo HILTUNEN; Peroxisomal multifunctional enzyme of β-oxidation metabolizing d-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization. Biochem J 1 January 1997; 321 (1): 21–28. doi: https://doi.org/10.1042/bj3210021
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