Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-β-lactamase family
Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-β-lactamase family
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DOI：
10.1074/jbc.m512359200
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发表时间：
2006-05-05
影响因子：
4.8
通讯作者：
Ueda, N
中科院分区：
文献类型：
--
作者：
Wang, J;Okamoto, Y;Ueda, N
在动物组织中，包括内源性大麻素anandamide的生物活性N-酰基乙醇胺通过属于金属-β-内酰胺酶家族的特异性磷脂酶D（NAPE-PLD）的催化从其相应的N-酰基磷脂酰乙醇胺（NAPE）形成。尽管其潜在的生理重要性，NAPE-PLD尚未被表征与纯化的酶制剂。在本研究中，我们在大肠杆菌中表达了重组NAPE-PLD，并高度纯化了它。纯化的酶被显着激活，在一个剂量依赖性的方式由毫摩尔浓度的Mg 2+以及Ca 2+，因此，似乎是组成性的活性。该酶表现出极高的特异性NAPE之间的各种甘油磷脂，但没有表现出明显的选择性不同的长链或中链N-酰基物种的NAPE。这些结果表明NAPE-PLD降解不同NAPE而不损伤其他膜磷脂的能力。金属分析揭示了催化重要的锌在NAPE PLD的存在。此外，定点诱变研究涉及金属β-内酰胺酶家族中高度保守的NAPE-PLD的几个组氨酸和天冬氨酸残基。Asp-147、His-185、His-187、Asp-189、His-190、His-253、Asp-284和His-321的单突变导致催化活性的消除或显著降低。此外，当6个半胱氨酸残基单独突变为丝氨酸时，只有C224 S显示出显著降低的活性。L207 F和H380 R的单核苷酸多态性也很低。因此，NAPE-PLD似乎通过与该家族的良好表征的成员相似的机制起作用，但在动物组织的脂质代谢中发挥独特的作用。
In animal tissues, bioactive N-acylethanolamines including the endocannabinoid anandamide are formed from their corresponding N-acylphosphatidylethanolamines (NAPEs) by the catalysis of a specific phospholipase D (NAPE-PLD) that belongs to the metallo-beta-lactamase family. Despite its potential physiological importance, NAPE-PLD has not yet been characterized with a purified enzyme preparation. In the present study we expressed a recombinant NAPE-PLD in Escherichia coli and highly purified it. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg2+ as well as Ca2+ and, hence, appeared to be constitutively active. The enzyme showed extremely high specificity for NAPEs among various glycerophospholipids but did not reveal obvious selectivity for different long chain or medium chain N-acyl species of NAPEs. These results suggested the ability of NAPE-PLD to degrade different NAPEs without damaging other membrane phospholipids. Metal analysis revealed the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies were addressed to several histidine and aspartic acid residues of NAPE-PLD that are highly conserved within the metallo-beta-lactamase family. Single mutations of Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 caused abolishment or remarkable reduction of the catalytic activity. Moreover, when six cysteine residues were individually mutated to serine, only C224S showed a considerably reduced activity. The activities of L207F and H380R found as single nucleotide polymorphisms were also low. Thus, NAPE-PLD appeared to function through a mechanism similar to those of the well characterized members of this family but play a unique role in the lipid metabolism of animal tissues.