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

N-花生四烯基乙醇胺是由动物组织中的甘油磷脂通过两步酶反应形成的，是大麻素受体的内源性配体。然而，相关酶的特性仍然很差，很难用分子生物学的方法来研究ANANDIAME的生物合成。最近，我们首次成功地克隆并功能表达了一种新的哺乳动物磷脂酶D型酶(NAPE-PLD)，它能从相应的N-酰基磷脂酰乙醇胺(NAPE)中产生ANAND胺和其他N-酰基乙醇胺(Okamoto等人)。J.Biol.化学。279、5298-305、2004年)。在本研究中，我们主要鉴定了重组大鼠NAPE-PLD。重组NAPE-PLD在大肠杆菌中以GST-融合蛋白的形式与分子伴侣一起表达。然后用CHAPS溶解该酶，然后用谷胱甘肽亲和层析和羟基磷灰石层析纯化得到明显的均一。纯化后的酶与NAPEs结合活性高，与C4-C&lt；20&gt；相反，该酶几乎与主要的膜甘油磷脂(磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸和磷脂酰肌醇)一起失活。这些结果表明，NAPE-PLD能够在不破坏其他膜磷脂的情况下特异性地降解不同的NAPE。纯化后的酶被毫摩尔浓度的Mg~(2+)和Ca~(2+)以剂量依赖方式激活。原子吸收光谱分析表明，NAPE-PLD中存在重要的催化活性锌。此外，定点突变研究表明，在金属内酰胺酶家族中高度保守的NAPE-PLD的Asp-147、His-185、His-187、Asp-189、His-190、His-253、Asp-284和His-321在催化活性中起着至关重要的作用。

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.