Qin Y M, Poutanen M H, Helander H M, Kvist A P, Siivari K M, Schmitz W, Conzelmann E, Hellman U, Hiltunen J K
Biocenter Oulu, University of Oulu, Finland.
Biochem J. 1997 Jan 1;321 ( Pt 1)(Pt 1):21-8. doi: 10.1042/bj3210021.
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 M(r) 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 M(r) of 79,331 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 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-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 beta-oxidation but through reciprocal stereochemical courses.
在本研究中，我们克隆并鉴定了一种名为perMFE-II的新型大鼠过氧化物酶体多功能酶（MFE）。从大鼠肝脏中纯化得到的分子量为31500的2-烯酰辅酶A水合酶2[马利拉、西瓦里、马克拉、亚洛宁、拉蒂帕、库瑙和希尔图宁（1993年）《生物化学杂志》268卷，21578 - 21585页]经胰蛋白酶裂解，所得肽段被分离并测序。令人惊讶的是，通过聚合酶链反应（PCR）扩增得到的全长互补脱氧核糖核酸（cDNA）具有一个2205碱基对的开放阅读框，编码一个预测分子量为79331的多肽，并且在其C末端（丙氨酸-赖氨酸-亮氨酸）含有一个潜在的过氧化物酶体靶向信号。水合酶2的测序肽段与cDNA推导的氨基酸序列中部完全匹配。预测的氨基酸序列与猪IV型17β-羟基类固醇脱氢酶以及酵母过氧化物酶体β-氧化的MFE具有高度相似性。重组的perMFE-II（在毕赤酵母中产生）具有2-烯酰辅酶A水合酶2和D特异性3-羟基酰基辅酶A脱氢酶活性，并且对几种直链反式-2-烯酰辅酶A、2-甲基十四碳烯酰辅酶A和 pristeneoyl-CoA酯具有催化活性。结果表明，除了先前描述的大鼠肝脏过氧化物酶体中的多功能异构酶-水合酶-脱氢酶（perMFE-I）外，大鼠肝脏过氧化物酶体中还存在另一种MFE。它们都催化β-氧化的连续水合酶和脱氢酶反应，但反应的立体化学过程相反。
