Shrestha Rhidaya, Dixon Richard A, Chapman Kent D
Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, Texas 76203, USA.
J Biol Chem. 2003 Sep 12;278(37):34990-7. doi: 10.1074/jbc.M305613200. Epub 2003 Jun 24.
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-酰基乙醇胺（NAEs）是植物和动物组织的内源性成分，在脊椎动物中，它们的水解终止了其作为脂质介质在内源性大麻素信号系统中的参与。负责哺乳动物体内NAE水解的膜结合酶已在分子水平上得到鉴定（命名为脂肪酸酰胺水解酶，FAAH），尽管在棉花幼苗的微粒体中鉴定出了类似的酶活性，但关于该酶在植物中的分子信息尚无报道。在此，我们报告了拟南芥FAAH同源物的鉴定、异源表达（在大肠杆菌中）及其生化特性。在植物基因组数据库中鉴定出含有特征性酰胺酶特征序列（PS00571）的拟南芥候选DNA序列，并使用拟南芥基因组序列设计合适的寡核苷酸引物，通过逆转录酶-PCR分离出一个cDNA。对该cDNA进行测序，预测其编码一个607个氨基酸的蛋白质，在酰胺酶特征结构域内与大鼠FAAH具有37%的同一性（全长范围内为18%）。已确定对FAAH催化重要的残基在拟南芥和大鼠蛋白质序列之间是保守的。此外，在拟南芥蛋白质序列中预测到靠近N端有一个单一的跨膜结构域，类似于大鼠FAAH蛋白质的跨膜结构域。推定的植物FAAH cDNA在大肠杆菌中表达为表位/组氨酸标签融合蛋白，并在非离子洗涤剂十二烷基麦芽糖苷中从细胞裂解物中溶解出来。亲和纯化的重组蛋白确实具有水解多种天然存在的N-酰基乙醇胺类型活性。重组拟南芥蛋白的动力学参数和抑制数据与先前在植物和动物系统中表征的该酶活性特性一致。这些数据共同在分子水平上为植物和动物之间NAEs代谢的保守机制提供了支持。
