Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-beta-lactamase family.
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.
Wang
Jun
J
Department of Biochemistry, School of Medicine, Kagawa University, Miki, Japan.
Okamoto
Yasuo
Y
Morishita
Jun
J
Tsuboi
Kazuhito
K
Miyatake
Akira
A
Ueda
Natsuo
N
D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D001483:Base Sequence; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D063388:Endocannabinoids; D004926:Escherichia coli / Q000378:metabolism; D020404:Glycerophospholipids / Q000737:chemistry; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000737:chemistry; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D051381:Rats; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D063388:Endocannabinoids; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D053284:Polyunsaturated Alkamides; D010739:Phospholipase D; D001618:beta-Lactamases; C078814:anandamide