Orientador: Carlos Rodrigues Brandão / Tese (doutorado) - Univesidade Estadual de Campinas, Instituto de Filosofia e Ciencias Humanas / Made available in DSpace on 2018-07-25T07:48:03Z (GMT). No. of bitstreams: 1 Hoefel_JoaoLuizdeMoraes_D.pdf: 6539002 bytes, checksum: 679c9f2359a040f9d7326bd39a05829f (MD5) Previous issue date: 1999 / Doutorado

Fatty Acid Amide Hydrolase (FAAH), a serine hydrolase family protein, hydrolyzes N-acylethanolamines (NAEs) by cleaving the amide bond linking the acyl group with ethanolamine to produce free fatty acids. Highly conserved ‘Amidase Signature (AS)’ sequence rich in serine, glycine and alanine residues characterize the protein. FAAH plays role in various physiological processes by regulating NAE levels, such as seedling growth, defense response. Understanding of the role of NAEs and FAAH has been however, limited to model plant Arabidopsis. Here, with interest to understand the role of FAAH in modulating NAE composition, tomato was chosen as a model system. Recently, SlFAAH1, an ortholog of AtFAAH1 was identified in tomato and was successfully expressed in prokaryotic expression system. Protein assay with lysate of cells expressing recombinant putative SlFAAH1 showed the ability to hydrolyze a polyunsaturated NAE (NAE20:4). Currently, additional assays are being carried out to determine optimal pH, temp, substrate specificity and associated enzyme kinetics. In parallel, the effect of exogenous NAEs on SlFAAH1 expression levels and during seedling development is being evaluated. Together, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development, and further allows for comparison with Arabidopsis and mammalian FAAH to determine its functional conservation.

N-acylethanolamines (NAEs) are fatty acid derivatives that occur naturally in plant and animal systems. In mammals, they regulate physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, and cell proliferation. NAEs are metabolized by fatty acid amide hydrolase (FAAH), which belongs to the amidase signature family. It is hypothesized that putative FAAH functions as the catalyst in the metabolism of N-acylethanolamine in tomato plants. To test the hypothesis, FAAH protein homologs were identified in tomato via in silico analysis. Among the six homologs identified, FAAH1 and FAAH2 were selected for further validation. This study is focused on 1) in silico analyses of SlFAAH2, 2) quantification of transcript levels for SlFAAH2, 3) determination of FAAH activity at various developmental stages of tomato, and 4) isolation of and synthesis of SlFAAH2 cDNA for cloning. Putative SlFAAH2 showed high homology to Arabidopsis FAAH1. Transcript levels, as measured by qPCR using RNA extracted from various developmental stages, were highest at 0 days and lowest at 4 days. Enzyme activity at certain developmental stages coincided with SlFAAH2 transcript levels. In order to confirm that putative SlFAAH2 encodes for an enzyme that hydrolyzes NAEs, SlFAAH2 gene was isolated from total RNA of tomato, cDNA was synthesized by reverse transcription and the gene was amplified by PCR for further cloning in a heterologous expression system for biochemical characterization. To gain better molecular and biochemical understanding of FAAH and determine its broader functions, it is pertinent to characterize FAAH in other plant species.