Characterization of Fatty Acid Amide Hydrolase in Physcomitrella Patens

Document Type

Presentation

Publication Date

4-9-2017

Description

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5‟- and 3‟-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with N-terminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

Location

Orlando, FL

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