Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation
Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation
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DOI：
10.1111/febs.13696
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发表时间：
2016-05-01
期刊：
影响因子：
5.4
通讯作者：
Brumer, Harry
中科院分区：
文献类型：
--
作者：
Attia, Mohamed;Stepper, Judith;Brumer, Harry
杂多糖木葡聚糖 (XyG) 占陆地植物细胞壁总碳水化合物含量的四分之一，因此代表了全球碳循环中的重要储库。 XyG 的复杂组成需要主链裂解内切木葡聚糖酶和侧链裂解外切糖苷酶的联合体才能完成糖化。尽管最近对相关侧链裂解外切糖苷酶进行了表征，但模型革兰氏阴性土壤腐生细菌日本细胞弧菌利用 XyG 的生化基础尚不完全清楚。我们提出了由基因位点 CJA_2477 编码的多模块酶的详细功能和结构特征。 CJA_2477 基因产物包含一个 N 端糖苷水解酶家族 74 (GH74) 内切木葡聚糖酶模块，以及来自家族 10 和 2（CBM10 和 CBM2）的两个碳水化合物结合模块 (CBM)。 GH74 催化结构域通过内解离作用模式为下游酶生成基于 Glc4 的低聚木葡萄糖 (XyGO) 底物。 GH74 模块的 X 射线晶体学，无论是单独的还是与跨越整个活性位点的 XyGO 产品复合，都揭示了专门适合 XyG 识别的广泛底物结合裂缝，该裂缝由 GH74 家族特征的两个七叶片螺旋桨域组成。附加的 CBM10 和 CBM2 成员明显不结合 XyG，也不结合其他可溶性多糖，而是特定的纤维素结合模块。总而言之，这些数据揭示了日本刺参利用木葡聚糖的第一步，并扩展了用于选择性生物量分析和利用的 GH 和 CBM 的库。
The heteropolysaccharide xyloglucan (XyG) comprises up to one-quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone-cleaving endoxyloglucanases and side-chain cleaving exo-glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram-negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side-chain cleaving exo-glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N-terminal glycoside hydrolase family 74 (GH74) endo-xyloglucanase module in train with two carbohydrate-binding modules (CBMs) from families 10 and 2 (CBM10 and CBM2). The GH74 catalytic domain generates Glc4-based xyloglucooligosaccharide (XyGO) substrates for downstream enzymes through an endo-dissociative mode of action. X-ray crystallography of the GH74 module, alone and in complex with XyGO products spanning the entire active site, revealed a broad substrate-binding cleft specifically adapted to XyG recognition, which is composed of two seven-bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose-binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization.