Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Laboratory for Immunogenomics, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan; and.
J Lipid Res. 2012 Oct;53(10):2242-2251. doi: 10.1194/jlr.D028951. Epub 2012 Jul 15.
Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.
内糖苷脂酶 (EGCase) 是一种糖苷酶，能够水解糖脂 (GSL) 中寡糖和神经酰胺之间的 β -糖苷键。在马红球菌（以前称为马红球菌 M-750）的培养液中发现了三种特异性不同的 EGCase 分子种类，分别命名为 EGCase I、II 和 III。本研究描述了 EGCase I 的分子克隆和重组酶的特性，该酶在红球菌表达系统中使用红球菌 erythropolis 高度表达。动力学分析显示，重组 EGCase I 的周转数 (k(cat))（k(cat)) 分别比 EGCase II 对 GM1a 和 Gb3Cer 的 k(cat)) 高 22 倍和 1200 倍，尽管两种酶对这些底物的 K(m)几乎相同。EGCase I（模型）和 EGCase II（晶体）的三维结构比较表明，一个柔性环悬挂在 EGCase II 的催化裂缝上，但不在 EGCase I 上。从 EGCase II 中删除该环增加了突变酶的 k(cat)，表明该环是影响催化区域中底物和产物周转率的关键因素。与 EGCase II 相比，重组 EGCase I 表现出广泛的特异性和良好的反应效率，使其非常适合 GSL 的综合分析。
