Shrestha Rhidaya, Kim Sang-chul, Dyer John M, Dixon Richard A, Chapman Kent D
University of North Texas, Center for Plant Lipid Research, Department of Biological Sciences, Denton, P.O. Box 305220, TX 76203, USA.
Biochim Biophys Acta. 2006 Mar;1761(3):324-34. doi: 10.1016/j.bbalip.2006.03.004. Epub 2006 Mar 31.
Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an "endocannabinoid" signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was identified which catalyzed the hydrolysis of NAEs in vitro suggesting a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, we provide evidence to support this concept by identifying candidate FAAH genes in monocots (Oryza sativa) and legumes (Medicago truncatula), which have similar, but not identical, exon-intron organizations. Corresponding M. truncatula and rice cDNAs were isolated and cloned into prokaryotic expression vectors and expressed as recombinant proteins in Escherichia coli. NAE amidohydrolase assays confirmed that these proteins indeed catalyzed the hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH, but not identical. Sequence alignments and motif analysis of plant FAAH enzymes revealed a conserved domain organization for these members of the amidase superfamily. Five amino-acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the FAAH sequences of six plant species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal structure as a template revealed a conserved protein core that formed the active site of each enzyme. Collectively, these results indicate that plant and mammalian FAAH proteins have similar structure/activity relationships despite limited overall sequence identity. Defining the molecular properties of NAE amidohydrolase enzymes in plants will help to better understand the metabolic regulation of NAE lipid mediators.
脂肪酸酰胺水解酶（FAAH）在调节脊椎动物体内内源性N-酰基乙醇胺（NAE）水平方面发挥着核心作用，并且在一定程度上构成了一条“内源性大麻素”信号通路，该通路调节动物体内多种生理和行为过程。最近，一种拟南芥FAAH同源物被鉴定出来，它在体外催化NAE的水解，这表明植物中存在一条由FAAH介导的内源性NAE代谢途径。在此，我们通过在单子叶植物（水稻）和豆科植物（蒺藜苜蓿）中鉴定候选FAAH基因来提供证据支持这一概念，这些基因具有相似但不完全相同的外显子-内含子结构。分离出蒺藜苜蓿和水稻相应的cDNA，并将其克隆到原核表达载体中，在大肠杆菌中表达为重组蛋白。NAE酰胺水解酶分析证实，这些蛋白确实在体外催化了14C标记的NAE的水解。水稻FAAH的动力学参数和抑制特性与拟南芥和大鼠FAAH相似，但并不完全相同。对植物FAAH酶的序列比对和基序分析揭示了酰胺酶超家族这些成员的保守结构域组织。经确定，对大鼠FAAH催化作用重要的五个氨基酸残基在六种植物的FAAH序列中完全保守。以大鼠FAAH晶体结构为模板对植物FAAH蛋白进行同源建模，结果显示形成每种酶活性位点的保守蛋白核心。总体而言，这些结果表明，尽管植物和哺乳动物FAAH蛋白的整体序列一致性有限，但它们具有相似的结构/活性关系。明确植物中NAE酰胺水解酶的分子特性将有助于更好地理解NAE脂质介质的代谢调控。
