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J. Biol. Chem., Vol. 281, Issue 18, 12325-12335, May 5, 2006

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Functional Analysis of the Purified Anandamide-generating Phospholipase D as a Member of the Metallo--lactamase Family^*

Jun Wang, Yasuo Okamoto, Jun Morishita, Kazuhito Tsuboi, Akira Miyatake^¶, and Natsuo Ueda¹

From the Department of Biochemistry, School of Medicine and the ^¶Life Science Research Center, Kagawa University, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan and the Department of Anesthesiology, The First Affiliated Hospital, China Medical University, Shenyang 110001, China

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--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--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.

Received for publication, November 17, 2005 , and in revised form, March 2, 2006.

^* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and grants-in-aid from Ono Medical Research Foundation, Uehara Memorial Foundation, Medical Institute Union Foundation, Nankai Ikueikai, the Ichiro Kanehara Foundation, Japan Research Foundation for Clinical Pharmacology, Cayman Chemical (Ann Arbor, MI), and Kagawa University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-87-891-2102; Fax: 81-87-891-2105; E-mail: nueda{at}med.kagawa-u.ac.jp.

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