Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice
Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice
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DOI：
10.1111/j.1365-313x.2008.03569.x
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发表时间：
2008-09-01
期刊：
影响因子：
7.2
通讯作者：
Ng, Carl K. -Y.
中科院分区：
文献类型：
--
作者：
Pata, Mickael O.;Wu, Bill X.;Ng, Carl K. -Y.
鞘脂是一组结构多样的分子，基于在动物、真菌和植物细胞中发现的长链鞘氨醇碱。与动物和酵母中的情况相反，对植物中鞘脂种类的谱以及它们在介导细胞过程中所起的作用知之甚少。在这里，我们报告的克隆和植物神经酰胺酶的特性从水稻（水稻）。粳稻Nipponbare）。序列分析表明水稻神经酰胺酶（OsCDase）与哺乳动物中性神经酰胺酶相似。我们证明OsCDase是一个真正的神经酰胺酶在酵母双敲除突变体Δ ypc 1 Δ pac 1，缺乏酵母神经酰胺酶YPC 1 p和YDC 1 p的异源表达。OsCDase的生化特性表明，OsCDase具有经典的米氏动力学，最适pH为5.7 ~ 6.0。Ca ~（2+）、Mg ~（2+）、Mn ~（2+）、Zn ~（2+）对OsCD酶活性有促进作用，Fe ~（2+）对OsCD酶活性有抑制作用。OsCD酶似乎使用神经酰胺而不是植物神经酰胺作为底物。亚细胞定位结果表明OsCDase定位于内质网和高尔基体，表明这些细胞器是植物神经酰胺代谢的场所。
Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Delta ypc1 Delta ydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca2+, Mg2+, Mn2+ and Zn2+, but inhibited in the presence of Fe2+. OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants.