Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

Abstract

N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in Escherichia coli), and the biochemical characterization of an Arabidopsis thaliana FAAH homologue. Candidate Arabidopsis DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using Arabidopsis genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the Arabidopsis and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the Arabidopsis protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/ His-tagged fusion protein in E. coli and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring N-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant Arabidopsis protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.N-Acylethanolamines (NAEs) 1 are lipid mediators that are produced from the phospholipase D-mediated hydrolysis of Nacylphosphatidylethanolamines, a minor membrane lipid constituent of cellular membranes (1). In animal systems, anandamide (NAE 20:4) acts as an endogenous ligand for cannabinoid receptors and has varied physiological roles, such as the modulation of neurotransmission in the central nervous system (2). Anandamide also activates vanilloid receptors, functions as an endogenous analgesic (3), and appears to be involved in neuroprotection (4,5). In other tissues, NAEs have been implicated in immunomodulation (6), synchronization of embryo development (7), and induction of apoptosis (8). These endogenous bioactive molecules lose their signaling activity upon hydrolysis by fatty acid amide hydrolase (FAAH; see Ref. 9).In plants NAEs are present in substantial amounts in desiccated seeds (ϳ1 g g Ϫ1 fresh weight), and their levels decline after a few hours of imbibition (10). Ind...

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

“…This amidase-signature has earlier been described for enzymes from mammals (Patricelli and Cravatt 2000) and bacteria (Shin et al 2002). The A. thaliana amidase-signature family comprises seven different members each encoded by a different gene from which only two proteins, AMIl (Pollmann et al 2003) and a fatty acid amide hydrolase (FAAH; Shrestha et al 2003), were characterized for their enzymatic activity. The third isoform (At5g09420) seems to be localized in the outer mitochondrial membrane in complex with a protein translocase (Chew et al 2004) and a fourth one (At3gl7970) has been found associated with proteins of the chloroplast Toc-complex after protein cross-linking (Sohrt and Solí 2000).…”

“…Cloning and expression of [His] 6 -tagged amidases Recombinant fatty acid amide hydrolase (FAAH) was expressed in Escherichia coli TOP10 harboring the expression vector pTrcHis2 (Invitrogen) containing the full-length 7vL4//-cDNA, kindly provided by Dr. Rhidaya Shrestha and Dr. Kent D. Chapman, as described by Shrestha et al (2003). Hexahistidine-tagged amidase 1 (AMIl) was expressed in E. coli M15 harboring the vector pQE-30 (Qiagen) with a 2?amHI/Sa/I-integrated AMil-cDNA according to the manufacturer's protocol with the following exceptions: Bacterial cultures were incubated at 37°C until they reached an OD 600 of 0.85.…”

“…The mRNA-level of AMIl in this tissue seemed fairly constant in plants between 3 and 8 weeks after germination (Pollmann et al 2003). To obtain more detailed information and to compare the expression pattern of AMIl and the related isogene FAAH (Shrestha et al 2003), total RNA was extracted from different tissues of A. thaliana. As the expression level of AMIl was expected to be too low to be detected by Northern-blot analysis RT-PCR was used to analyze the specific expression patterns of the two genes in different organs.…”

Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N -acylethanolamines

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] linoleic acid (53 mCi⅐mmol Ϫ1 in ethanol; 1Ci ϭ 37 GBq) was purchased from PerkinElmer Life Sciences (Boston, MA), and [1-14 C] lauric acid (53 mCi⅐mmol Ϫ1 in ethanol) was from Amersham Pharmacia Biosciences (Piscataway, NJ). Specific types of NAEs were synthesized from respective radiolabeled free fatty acids as described (8).…”

“…We recently reported the molecular identification of a functional homologue of FAAH in Arabidopsis thaliana that converts a wide range of NAEs to their corresponding free fatty acids and ethanolamine (14). Functional homologues of the Arabidopsis FAAH (AtFAAH) also were identified in Oryza sativa and Medicago truncatula, supporting a common mechanism for the regulation of NAE hydrolysis in diverse plant species (15).…”