Functional analysis of a novel enzyme of the phospholipase D type involved in the endocannabinoid

Anandamide （N-arachidonoylethanolamine）是一种内源性大麻素受体（内源性大麻素）的配体，由膜甘油磷脂通过两步酶反应在动物组织中形成。然而，相关酶的特征尚不明确，用分子生物学方法研究菊芋酰胺的生物合成存在困难。最近，我们首次成功克隆了一种新的哺乳动物磷脂酶D型（NAPE-PLD）的cDNA，并成功表达了该酶的功能，该酶能从相应的n -酰基磷脂酰乙醇胺（nape）中生成anandamide和其他n -酰基乙醇胺（Okamoto et al.）。生物。化学，279,5298 - 305,2004)。在本研究中，我们主要表征了重组大鼠NAPE-PLD。重组NAPE-PLD在大肠杆菌中表达为gst融合蛋白，并与分子伴侣蛋白结合表达。然后用CHAPS溶解酶，然后用谷胱甘肽亲和层析和羟基磷灰石层析纯化到明显的均匀性。纯化后的酶对nape具有高活性，对C_4-C_<20>的n -酰基种类无明显区分。相反，该酶对主要的膜甘油磷脂（磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸和磷脂酰肌醇）几乎无活性。这些结果表明NAPE-PLD能够特异性地降解不同的nape而不破坏其他膜磷脂。纯化后的酶在毫摩尔浓度的Mg^<2+>和Ca^<2+>下呈剂量依赖性激活。原子吸收光谱分析表明，NAPE-PLD中存在具有重要催化作用的锌。此外，位点诱变研究表明，npe - pld的Asp-147、His-185、His-187、Asp-189、His-190、His-253、Asp-284和His-321是金属-□-内酰胺酶家族中高度保守的分子，在催化活性中起关键作用。

Anandamide (N-arachidonoylethanolamine), which was found as an endogenous ligand for cannabinoid receptors (an endocannabinoid), is formed from membrane glycerophospholipids by two-step enzyme reactions in animal tissues. However, the responsible enzymes remained poorly characterized, and it has been difficult to study the anandamide biosynthesis by molecular biological approaches. Recently we succeeded for the first time in cDNA cloning and functional expression of a novel mammalian enzyme of the phospholipase D type (NAPE-PLD) that generates anandamide and other N-acylethanolamines from their corresponding N-acylphosphatidylethanolamines (NAPEs) (Okamoto et al. J. Biol. Chem. 279, 5298-305, 2004). In the present study, we principally characterized recombinant rat NAPE-PLD.The recombinant NAPE-PLD was expressed in Escherichia coli as a GST-fusion protein together with molecular chaperone. The enzyme was then solubilized with CHAPS, followed by purification to apparent homogeneity by glutathione affinity chromatography and hydroxyapatite chromatography. The purified enzyme was highly active with NAPEs, and did not discriminate various N-acyl species with C_4-C_<20>. In contrast, the enzyme was almost inactive with major membrane glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol). These results suggested the ability of NAPE-PLD to specifically degrade different NAPEs without damaging other membrane phospholipids. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg^<2+> as well as Ca^<2+>. Atomic absorption spectrometry exhibited the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies revealed that Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 of NAPE-PLD, that are highly conserved within the metallo-□-lactamase family, play crucial roles in the catalytic activity.