PEROXISOMAL MULTIFUNCTIONAL ENZYME OF BETA-OXIDATION METABOLIZING D-3-HYDROXYACYL-COA ESTERS IN RAT-LIVER - MOLECULAR-CLONING, EXPRESSION AND CHARACTERIZATION

In the present study we have cloned and characterized a novel rat pero xisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-e noyl-CoA hydratase 2 with an M(r) of 31 500 from rat liver [Malila, Si ivari, Makela, Jalonen, Latipaa, Kunau and Hiltunen (1993) J. Biol. Ch em. 268, 21578-21585] was subjected to tryptic fragmentation and the r esulting peptides were isolated and sequenced. Surprisingly, the full- length cDNA, amplified by PCR, had an open reading frame of 2205 bp en coding a polypeptide with a predicted M(r) of 79331 and contained a po tential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in th e middle portion of the cDNA-derived amino acid sequence. The predicte d amino acid sequence showed a high degree of similarity with pig 17 b eta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) ha d 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogena se activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA ester s. The results showed that in addition to an earlier described multifu nctional isomerase-hydratase-dehydrogenase enzyme from rat liver perox isomes (perMFE-I), another MFE exists in rat liver peroxisomes, They b oth catalyse sequential hydratase and dehydrogenase reactions of beta- oxidation but through reciprocal stereochemical courses.