Biochemical Characterization of Fatty Acid Amide Hydrolase in Physcomitrella Patens

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N-acylethanolamines (NAEs) are a group of fatty acid ethanolamides and their metabolic pathway is highly conserved in eukaryotes. However, metabolites such as NAE 20:4 (anandamide) are known to occur in mammalian systems but not in higher plants. Anandamide is an endocannabinoid receptor ligand and mediates stress responses and regulates various physiological processes such as neuroprotection, pain perception and appetite suppression in animals. Interestingly anandamide occurrence was recently reported in a highly stress tolerant early land plant, Physcomitrella patens but its physiological role remains to be elucidated. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. To test this hypothesis, arabidopsis FAAH homolog was used to search moss database using BLASTP. Eight putative FAAH candidates (PpFAAH1-8), with an amidase signature sequence and conserved catalytic sites, were identified. Among these, PpFAAH1 and PpFAAH2 responded to exogenous NAE, and their 3D predicted protein structure closely resembled to that of AtFAAH1. The 1.8Kb coding region of putative PpFAAH1 was chosen for further characterization and was PCR amplified, cloned into TrcHis2 expression vector and transformed into E. coli TOP10 cells. Upon confirmation of the positive clones and induction of proteins, expressed proteins will be purified by Ni+2 affinity column chromatography, confirmed by western blot and analyzed for its substrate specificity using radiolabelled anandamide. Lipids extracted from reaction mixture will be separated by thin layer chromatography and detected by radiometric scanning. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.


Johnson City, TN

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