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

J Biol Chem. 2006 May 5;281(18):12325-35. doi: 10.1074/jbc.M512359200. Epub 2006 Mar 9.

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-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Arachidonic Acids
  • Calcium Channel Blockers
  • Endocannabinoids
  • Glycerophospholipids
  • Phosphatidic Acids
  • Polyunsaturated Alkamides
  • Phospholipase D
  • beta-Lactamases
  • anandamide