Peroxisomal multifunctional enzyme of β-oxidation metabolizing d -3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization
| Autor: | J K Hiltunen, K M Siivari, Ari-Pekka Kvist, Yong-Mei Qin, H M Helander, Matti Poutanen, Ulf Hellman, W Schmitz, Ernst Conzelmann |
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| Rok vydání: | 1997 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Dehydrogenase Molecular cloning Microbodies Polymerase Chain Reaction Biochemistry Gene Expression Regulation Enzymologic Pichia pastoris Peroxisomal Multifunctional Enzyme Type 2 Animals Amino Acid Sequence Cloning Molecular Enoyl-CoA Hydratase Molecular Biology Peptide sequence Beta oxidation Peroxisomal targeting signal Base Sequence biology Cell Biology Peroxisome Blotting Northern biology.organism_classification Molecular biology Rats Liver Sequence Alignment Research Article |
| Zdroj: | Biochemical Journal. 321:21-28 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj3210021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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