Cloning and characterization of the acid lipase from castor beans
Cloning and characterization of the acid lipase from castor beans
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DOI：
10.1074/jbc.m408686200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Eastmond, PJ
中科院分区：
文献类型：
--
作者：
Eastmond, PJ
蓖麻胚乳含有一种与油体膜相关的酸性脂肪酶活性。为了鉴定这种酶，对纯化的油体进行蛋白质组学分析。一个类似于60 kDa的蛋白质被鉴定（RcOBL 1），它与丝状真菌米黑根毛霉的脂肪酶具有同源性。RcOBL 1含有α/β-水解酶的特征，如假定的催化三联体（DH下的（S））和围绕亲核丝氨酸残基的保守五肽（XG下的GX（S））。RcOBL 1在大肠杆菌中异源表达，并显示在酸性pH（最适类似于4.5）下水解三油酸甘油酯。RcOBL 1可以水解一系列三酰甘油，但对磷脂没有活性。该活性对丝氨酸试剂二乙基对硝基苯基磷酸敏感，表明RcOBL 1是丝氨酸酯酶。针对RcOBL 1产生的抗体用于显示该蛋白质仅限于胚乳，在胚乳中其与油体的表面相关联。这是第一个证据的分子身份的油体相关脂肪酶从植物。序列比较显示，OBL 1样蛋白家族存在于许多物种中，并且它们可能在调节脂解中发挥重要作用。
Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A similar to60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an alpha/beta-hydrolase, such as a putative catalytic triad ((S) under bar DH) and a conserved pentapeptide ( GX (S) under bar XG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima similar to4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.