Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-beta-lactamase family.

Wang J, Okamoto Y, Morishita J, Tsuboi K, Miyatake A, Ueda N
J Biol Chem. 2006 281 (18): 12325-35

PMID: 16527816 · DOI:10.1074/jbc.M512359200

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.

MeSH Terms (17)

Amino Acid Sequence Animals Arachidonic Acids Base Sequence beta-Lactamases Calcium Channel Blockers Endocannabinoids Escherichia coli Glycerophospholipids Humans Mice Molecular Sequence Data Phosphatidic Acids Phospholipase D Polyunsaturated Alkamides Rats Sequence Homology, Amino Acid

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