Yaoi Katsuro, Kondo Hidemasa, Hiyoshi Ayako, Noro Natsuko, Sugimoto Hiroshi, Tsuda Sakae, Mitsuishi Yasushi, Miyazaki Kentaro
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
J Mol Biol. 2007 Jun 29;370(1):53-62. doi: 10.1016/j.jmb.2007.04.035. Epub 2007 Apr 19.
Oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH) is a unique exo-beta-1,4-glucanase that belongs to glycoside hydrolase family 74. The enzyme recognizes the reducing end of xyloglucan oligosaccharides and releases two glucosyl residue segments from the reducing end of the main chain. Previously, we reported that OXG-RCBH consists of two seven-bladed beta-propeller domains. There is a large cleft between the two domains, and a unique loop encloses one side of the active site cleft. Here, we report the X-ray crystal structure of the OXG-RCBH-substrate complex determined to a resolution of 2.4 A. The substrate bound to the cleft, and its reducing end was arranged near the loop region that is believed to impart OXG-RCBH with its activity. We constructed a deletion mutant of the loop region and conducted a detailed analysis. A deletion mutant of the loop region showed endo-activity with altered substrate recognition. More specifically, cleavage occurred randomly instead of at specific sites, most likely due to the misalignment of the substrate within the subsite. We believe that the loop imparts unique substrate specificity with exo-mode hydrolysis in OXG-RCBH.
寡聚木葡聚糖还原端特异性纤维二糖水解酶（OXG-RCBH）是一种独特的外切β-1,4-葡聚糖酶，属于糖苷水解酶家族74。该酶识别木葡聚糖寡糖的还原端，并从主链的还原端释放两个葡萄糖基残基片段。此前，我们报道OXG-RCBH由两个七叶β-螺旋桨结构域组成。两个结构域之间有一个大裂缝，一个独特的环包围着活性位点裂缝的一侧。在此，我们报道了分辨率为2.4 Å的OXG-RCBH-底物复合物的X射线晶体结构。底物结合在裂缝中，其还原端排列在据信赋予OXG-RCBH活性的环区域附近。我们构建了环区域的缺失突变体并进行了详细分析。环区域的缺失突变体表现出内切活性，底物识别发生改变。更具体地说，切割随机发生而不是在特定位点，这很可能是由于底物在亚位点内的错位。我们认为，该环赋予OXG-RCBH以外切模式水解的独特底物特异性。
