Biochemical Characterization of Fatty Acid Amide Hydrolase in Physcomitrella Patens

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N-acylethanolamines (NAEs) are fatty acid ethanolamides that mediate stress responses in plants and animals. NAEs such as NAE 20:4 (anandamide) have only been reported in mammals and they regulate processes like neuroprotection and pain perception. Interestingly, we discovered the unique occurrence of anandamide in moss, Physcomitrella patens, a stress tolerant early land plant. 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. I specifically propose to biochemically characterize FAAH enzyme that degrades anandamide. For this, Arabidopsis FAAH (AtFAAH) homolog was identified in moss database using BLASTP. The predicted protein structure of putative moss FAAH (PpFAAH) closely resembled to that of AtFAAH with conserved amidase signature sequence and catalytic triad residues: Lys205, Ser281, Ser305. Transcript levels of PpFAAH increased five-fold when moss was grown on excess NAE containing media. PpFAAH cDNA was PCR amplified and cloned into pET23a expression vector and transformed into RIL E. coli cells and confirmed by colony PCR. Heterologously expressed protein will be purified by Ni+2 affinity column chromatography and confirmed by western blot using anti-His-tag antibody. For biochemical characterization, enzyme will be presented with 14C NAE 20:4 substrate and rate of product free fatty acid formed will be quantified by extracting lipids from reaction mixture and separating by thin layer chromatography followed by radiometric scanning. E. coli cells expressing AtFAAH enzyme will be used as control. A complete characterization of the PpFAAH enzyme will be carried out to determine the kinetics, optimal temperature and pH conditions. 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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