Shiojima Yuta, Sano Ryotaro, Kozono Takuma, Nishikawa Atsushi, Kojima Yuka, Yoshida Makoto, Sunagawa Naoki, Igarashi Kiyohiko, Tonozuka Takashi
1 Department of Applied Biological Science, Tokyo University of Agriculture and Technology.
2 Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology.
J Appl Glycosci (1999). 2025 May 20;72(2):7202104. doi: 10.5458/jag.7202104. eCollection 2025.
Glycoside hydrolase family 131 (GH131) proteins are found in oomycetes, ascomycetes, and basidiomycetes, and have been reported to hydrolyze various β-glucan polysaccharides. , a model basidiomycete, contains two GH131 proteins, CcGH131A and CcGH131B. This study focuses on the structural and functional properties of CcGH131B, a protein that lacks the carbohydrate bonding module 1 (CBM1) domain present in CcGH131A. The crystal structure of CcGH131B was determined. The structure displayed a β-jelly roll fold with extra loops and α-helices, resulting in a deeper substrate-binding groove compared to CcGH131A and also PaGluc131A, a GH131 protein from . A cellobiose-bound structure of the E161A mutant, in which the potential catalytic residue Glu161 was substituted with Ala, showed that the region of the minus subsites bind cellulose. In contrast, the region of the plus subsites mainly consists of hydrophobic amino acid residues and appeared to interact with hydrophobic molecules rather than with carbohydrates. Analysis using native affinity polyacrylamide gel electrophoresis showed that CcGH131B interacted with cellulosic polysaccharides such as methylcellulose and carboxymethylcellulose, while the protein exhibited no detectable enzymatic activity under the tested conditions. These results suggest that the substrate specificity of CcGH131B is likely to be different from those of CcGH131A and PaGluc131A.
糖苷水解酶家族131（GH131）蛋白存在于卵菌纲、子囊菌纲和担子菌纲中，据报道可水解各种β-葡聚糖多糖。作为一种典型的担子菌，含有两种GH131蛋白，即CcGH131A和CcGH131B。本研究聚焦于CcGH131B的结构和功能特性，该蛋白缺乏CcGH131A中存在的碳水化合物结合模块1（CBM1）结构域。测定了CcGH131B的晶体结构。该结构呈现出具有额外环和α-螺旋的β-果冻卷折叠，与CcGH131A以及来自[具体物种未提及]的GH131蛋白PaGluc131A相比，产生了更深的底物结合槽。E161A突变体的纤维二糖结合结构（其中潜在的催化残基Glu161被Ala取代）表明，负亚位点区域结合纤维素。相比之下，正亚位点区域主要由疏水氨基酸残基组成，似乎与疏水分子相互作用而非与碳水化合物相互作用。使用天然亲和聚丙烯酰胺凝胶电泳分析表明，CcGH131B与甲基纤维素和羧甲基纤维素等纤维素多糖相互作用，而该蛋白在测试条件下未表现出可检测到的酶活性。这些结果表明，CcGH131B的底物特异性可能与CcGH131A和PaGluc131A不同。
