Identification and Characterization of N-Acylethanolamine Hydrolyzing Enzyme in Solanum Lycopersicum
N-acylethanolamines (NAEs) are fatty acid ethanolamides that occur in a wide range of eukaryotes and their composition is specific to species, developmental stage and tissue type. In plants, NAEs negatively regulate growth and mediate stress responses. The function of NAEs is terminated by a highly conserved enzyme fatty acid amide hydrolase (FAAH). In plants, FAAH homologue has been characterized only for model plant Arabidopsis. Under normal growth conditions, AtFAAH overexpressor seedlings showed enhanced growth suggesting that FAAH might act as a modulator of endogenous NAE levels and regulate growth. Because of the significant role NAEs were shown to play in model plant Arabidopsis, it is pertinent to elucidate this conserved metabolic pathway in crop species such as tomato (Solanum lycopersicum) that may lead to improved productivity. We hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. With AtFAAH as a template and using BLASTX, we identified two putative FAAH sequences in tomato. CLUSTALW alignment showed conserved amidase signature sequence and the catalytic triad. Molecular visualization system (PyMOL) revealed that the protein structures of putative SlFAAH1and 2 were similar in domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. This research is expected to lead to characterization of NAE pathway in a crop plant. Long-term implications of this study include development of molecular and biochemical tools necessary to improve tolerance to abiotic stress and increase crop productivity.
Stuffle, Derek; Tiwari, Vijay; and Kilaru, Aruna. (false). 2016. Identification and Characterization of N-Acylethanolamine Hydrolyzing Enzyme in Solanum Lycopersicum. The 30th Anniversary National Conference on Undergraduate Research, Asheville, NC. https://apps.cur.org/ncur2018/archive/Display_NCUR.aspx?id=95561