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

Abstract

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 Mg 2؉ as well as Ca 2؉ 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, sitedirected 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.

An Important Role for N-Acylethanolamine Acid Amidase in the Complete Freunds Adjuvant Rat Model of Arthritis

“…First, an N-acyltransferase activity, which remains to be molecularly cloned, transfers the palmitic or oleic acid chain from the sn-1 position of phosphatidylcholine to the amino group of phosphatidylethanolamine (PE). The newly formed N-acyl-PE is hydrolyzed to PEA and OEA by a selective phospholipase D, whose primary sequence and tridimensional structure have been elucidated (Okamoto et al, 2004;Wang et al, 2006;Magotti et al, 2015). Two distinct enzymes, fatty acid amide hydrolase and NAAA, are able to catalyze the hydrolysis of PEA and OEA into their constituent fatty acids and ethanolamine.…”

Cannabinoids and the Urinary Bladder

“…Initial characterization of NAPE-PLD revealed the enzyme to be membrane associated and it lacks the ability to catalyze a transphosphatidylation reaction, which is a common feature of known PLDs [22]. NAPE-PLD is the first PLD-type phosphodiesterase which belongs to the metallo-β-lactamase family [23]. Unlike classical neurotransmitters and neuropeptides, its primary product, AEA is not stored in vesicles but synthesized and released "on demand" in response to physiological and pathological stimuli, hormones neurotransmitters and depolarizing agents from its direct biosynthetic precursor N-arachidonoylphosphatidylethanolamine (NAPE) a phospholipid commonly found in biological membranes [24,25].…”

Isolevuglandin-modified phosphatidylethanolamine is metabolized by NAPE-hydrolyzing phospholipase D

“…The assay was analyzed by detecting the levels of NBD-phosphatidic acid (PA) using fl uorescencecoupled HPLC (460 nm excitation, 534 nm emission). Samples were injected onto a C18 column (Grace Alltech; 4.6 mm × 150 mm, 5 ) and products separated using a 34 min gradient from 40% solvent 1 (1 mM ammonium acetate in water) to 94% solvent 2 (1 mM ammonium acetate in reagent alcohol) with a C12:0NAPE having the highest hydrolysis rate ( 15 …”