Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum.
BACKGROUND
Anandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide.


RESULTS
A putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates.


CONCLUSIONS
This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.
National Research Council, Institute for Biological Sciences, 100, Sussex Drive, Ottawa, ON K1A 0R6, Canada.
Dhamodharan
D
Neelamegan
Ian C
IC
Schoenhofen
James C
JC
Richards
Andrew D
AD
Cox
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001095:Arachidonic Acids / Q000378:metabolism*; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D063388:Endocannabinoids / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D053284:Polyunsaturated Alkamides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D018076:DNA, Complementary; D063388:Endocannabinoids; D008565:Membrane Proteins; D053284:Polyunsaturated Alkamides; D011994:Recombinant Proteins; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide