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Functional Characterization and Structural Basis of the β-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-07-11 , DOI: 10.1021/acs.jafc.2c03410 Xin Fu 1 , Xin-Yi Zan 1 , Lei Sun 1 , Ming Tan 1 , Feng-Jie Cui 1, 2 , Ying-Ying Liang 1 , Li-Juan Meng 1 , Wen-Jing Sun 1, 2
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-07-11 , DOI: 10.1021/acs.jafc.2c03410 Xin Fu 1 , Xin-Yi Zan 1 , Lei Sun 1 , Ming Tan 1 , Feng-Jie Cui 1, 2 , Ying-Ying Liang 1 , Li-Juan Meng 1 , Wen-Jing Sun 1, 2
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β-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membrane-bound β-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/μg, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a Km value of 84.28 μM at pH 7.0 and synthesized β-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDP-glucose potentially bound to at least 11 residues via hydrogen bonds, π-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of β-1,3-glucan synthases in mushrooms.
中文翻译：
北冬虫夏草β-1,3-葡聚糖合酶CMGLS的功能表征和结构基础
β-1,3-葡聚糖合酶在葡聚糖合成、细胞壁组装和真菌生长中起关键作用。然而，它们的多跨膜结构域（超过 14 个 TMH）和大分子量（超过 100 kDa）显着阻碍了对其催化特性和机制的理解。在本研究中，克隆、鉴定和分析了北虫草中编码 221.7 kDa 膜结合 β-1,3-葡聚糖合酶 CMGLS的 5841-bp 基因CMGLS 。CMGLS部分纯化，比活为87.72 pmol/min/μg，纯化倍数为121，采用产物包埋法纯化，收率为10.16%。CMGLS 对 UDP-葡萄糖具有严格的特异性，K m在 pH 7.0 时的值为 84.28 μM，并合成了最大聚合度 (DP) 为 70 的 β-1,3-葡聚糖。在 AlphaFold 和分子对接的帮助下，CMGLS 的 3D 结构及其与底物 UDP- 的结合特征据我们所知，葡萄糖首次被提出。UDP-葡萄糖可能通过氢键、π-堆积和盐桥与至少 11 个残基结合，Arg 1436 被预测为直接与 UDP-葡萄糖上的葡萄糖、磷酸盐和核糖环部分相互作用的关键残基。这些发现将为认识和了解蘑菇中β-1,3-葡聚糖合酶的葡聚糖合成过程和催化机制开辟一条途径。
更新日期：2022-07-11
中文翻译：
北冬虫夏草β-1,3-葡聚糖合酶CMGLS的功能表征和结构基础
β-1,3-葡聚糖合酶在葡聚糖合成、细胞壁组装和真菌生长中起关键作用。然而，它们的多跨膜结构域（超过 14 个 TMH）和大分子量（超过 100 kDa）显着阻碍了对其催化特性和机制的理解。在本研究中，克隆、鉴定和分析了北虫草中编码 221.7 kDa 膜结合 β-1,3-葡聚糖合酶 CMGLS的 5841-bp 基因CMGLS 。CMGLS部分纯化，比活为87.72 pmol/min/μg，纯化倍数为121，采用产物包埋法纯化，收率为10.16%。CMGLS 对 UDP-葡萄糖具有严格的特异性，K m在 pH 7.0 时的值为 84.28 μM，并合成了最大聚合度 (DP) 为 70 的 β-1,3-葡聚糖。在 AlphaFold 和分子对接的帮助下，CMGLS 的 3D 结构及其与底物 UDP- 的结合特征据我们所知，葡萄糖首次被提出。UDP-葡萄糖可能通过氢键、π-堆积和盐桥与至少 11 个残基结合，Arg 1436 被预测为直接与 UDP-葡萄糖上的葡萄糖、磷酸盐和核糖环部分相互作用的关键残基。这些发现将为认识和了解蘑菇中β-1,3-葡聚糖合酶的葡聚糖合成过程和催化机制开辟一条途径。
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