Biochemical Characterization of Tomato Fatty Acid Amide Hydrolase

Document Type

Presentation

Publication Date

4-11-2017

Description

N-acylethanolamines (NAEs) are present in wide range of organisms and belong to family of functionally diverse signaling lipids. They consist of a fatty acid with varying chain lengths and ethanolamine linked via an amide bond. The level of NAEs is modulated by their hydrolysis to ethanolamine and fatty acid by an enzyme fatty acid amide hydrolase (FAAH). FAAH is an integral membrane protein that belongs to “amidase signature” superfamily of proteins, which is characterized by highly conserved region rich in serine, glycine and alanine. FAAH directly or indirectly plays a role in modulation of various physiological processes by regulating NAE levels. Although the role of NAEs and its key modulator FAAH has been studied in other plants, their role in tomato model is limited and unknown. More recently, SlFAAH1, an ortholog of AtFAAH1, was identified in tomato and cloned into bacterial expression system. However, putative SlFAAH1 function and distinct features are yet to be determined. It is hypothesized that the putative SlFAAH1 catalyzes the hydrolysis of NAEs and modulates the level of NAEs during the seedling development in tomato. To this extent, a putative SlFAAH1 (previously identified and cloned in pET-23a vector) will be biochemically characterized and also effect of NAEs on seedling development will be studied. Thus far, SlFAAH1 cloned in pET-23a vector was expressed in RIL cell line (prokaryotic expression system) followed by conformation of positive transformant by colony PCR. Currently, protein expression and confirmation of SlFAAH in the positive transformant is being done. The expressed protein will be characterized for its hydrolytic activity using radiolabelled substrate. The effect of exogenous NAEs during seedling development will be studied with regards to expression level of SlFAAH1 by qPCR and composition of NAE during the seedling development to determine the role of NAE during seedling development. Thus, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development. Longterm studies will identify the significance of highly conserved NAE pathway in eukaryotes.

Location

Johnson City, TN

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