N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: a novel enzyme of the beta-lactamase fold family releasing anandamide and other N-acylethanolamines.
N-acylethanolamines (NAEs) are a lipid class present in brain and other animal tissues and contains anandamide (an endocannabinoid) and other bioactive substances. NAEs are formed from N-acylphosphatidylethanolamines (NAPEs) by a phospholipase D (PLD)-type enzyme abbreviated to NAPE-PLD. Although this enzyme has been recognized for more than 20 years, its molecular cloning has only recently been achieved by us. We highly purified NAPE-PLD from the particulate fraction of rat heart, and on the basis of peptide sequences with the purified enzyme cloned its cDNA from mouse, rat and human. The deduced primary structures revealed no homology with any PLDs so far reported, but was suggested to belong to the beta-lactamase fold family. When overexpressed in COS-7 cells, the NAPE-PLD activity increased about 1000-fold in comparison with the endogenous activity. The recombinant enzyme generated various long-chain NAEs including anandamide from their corresponding NAPEs at similar rates. However, the enzyme was inactive with phosphatidylethanolamine and phosphatidylcholine and did not catalyze transphosphatidylation, a reaction characteristic of PLD. The enzyme was widely expressed in murine organs with higher levels in brain, testis and kidney. The existence of NAPE-PLD specifically hydrolyzing NAPEs to NAEs emphasizes physiological significance of NAEs including anandamide in brain and other tissues.
Ueda
Natsuo
N
Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan. nueda@med.kagawa-u.ac.jp
Okamoto
Yasuo
Y
Morishita
Jun
J
D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D063388:Endocannabinoids; D004983:Ethanolamines / Q000737:chemistry / Q000378:metabolism*; D040461:Gene Components; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013737:Testis / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001095:Arachidonic Acids; D018076:DNA, Complementary; D063388:Endocannabinoids; D004983:Ethanolamines; D010739:Phospholipase D; D053284:Polyunsaturated Alkamides; D011994:Recombinant Proteins; D001618:beta-Lactamases; C022203:N-acylethanolamines; C506062:Napepld protein, mouse; C078814:anandamide