Mechanistic Insights into the Hydrolysis and Synthesis of Ceramide by Neutral Ceramidase
Mechanistic Insights into the Hydrolysis and Synthesis of Ceramide by Neutral Ceramidase
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DOI：
10.1074/jbc.m808232200
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发表时间：
2009-04-03
影响因子：
4.8
通讯作者：
Ito, Makoto
中科院分区：
文献类型：
--
作者：
Inoue, Tsuyoshi;Okino, Nozomu;Ito, Makoto
神经酰胺酶（CDase; EC 3.5.1.23）水解神经酰胺以产生鞘氨醇和脂肪酸。该酶在真核生物中的多种生理事件中起调节作用，并且还在特定细菌中作为外毒素起作用。在2.2和1.4埃的分辨率，分别测定了C2-神经酰胺结合和非结合形式的铜绿假单胞菌（PaCD）的中性CD酶的晶体结构。PaCD由两个结构域组成，Zn ~（2+）和Mg ~（2+）/Ca ~（2+）结合位点分别位于N端结构域的中心和结构域之间的界面。C2-神经酰胺结合和未结合形式之间的结构比较揭示了C2-神经酰胺结合后环I发生的开-闭构象变化。在闭合状态下，该环位于Zn 2+配位位点上方和底物结合位点的开口上方。周围的Zn 2+的PaCD和大鼠中性CD酶的残基的突变分析表明，神经酰胺的N-酰基键的切割或创建遵循类似的机制，观察到的Zn 2+依赖性羧肽酶。研究结果为了解神经酰胺水解合成的分子机理提供了依据
Ceramidase (CDase; EC 3.5.1.23) hydrolyzes ceramide to generate sphingosine and fatty acid. The enzyme plays a regulatory role in a variety of physiological events in eukaryotes and also functions as an exotoxin in particular bacteria. The crystal structures of neutral CDase from Pseudomonas aeruginosa (PaCD) in the C2-ceramide-bound and -unbound forms were determined at 2.2 and 1.4 angstrom resolutions, respectively. PaCD consists of two domains, and the Zn2+- and Mg2+/Ca2+-binding sites are found within the center of the N-terminal domain and the interface between the domains, respectively. The structural comparison between the C2-ceramide-bound and unbound forms revealed an open-closed conformational change occurring to loop I upon binding of C2-ceramide. In the closed state, this loop sits above the Zn2+ coordination site and over the opening to the substrate binding site. Mutational analyses of residues surrounding the Zn2+ of PaCD and rat neutral CDase revealed that the cleavage or creation of the N-acyl linkage of ceramide follows a similar mechanism as observed for the Zn2+-dependent carboxypeptidases. The results provide an understanding of the molecular mechanism of hydrolysis and synthesis of ceramide