Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideumReport as inadecuate




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BMC Microbiology

, 12:124

First Online: 25 June 2012Received: 26 January 2012Accepted: 25 June 2012DOI: 10.1186-1471-2180-12-124

Cite this article as: Neelamegan, D., Schoenhofen, I.C., Richards, J.C.
et al.
BMC Microbiol 2012 12: 124.
doi:10.1186-1471-2180-12-124

Abstract

BackgroundAnandamide 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.

ResultsA 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.

ConclusionsThis 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.

AbbreviationsNAEsN-acylethanolamines

ASAmidase signature

FAAHFatty acid amide hydrolase

MAFPMethyl arachidonoyl fluorophosphonate

PMSFPhenylmethylsulfonyl fluoride

ApNAArachidonoyl p-nitroaniline

DpNADecanoyl p-nitroaniline

MBPMaltose binding protein

DMSODimethyl sulphoxide.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2180-12-124 contains supplementary material, which is available to authorized users.

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Author: Dhamodharan Neelamegan - Ian C Schoenhofen - James C Richards - Andrew D Cox

Source: https://link.springer.com/



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