Abstract：
Agarans are sulfated galactans extracted from red algae with high structural complexity, of which natural methylation often occurs on the O-6 position of its β-d-galactopyranose units. Although many agaran degrading enzymes, including agarases and porphyranases, have been characterized, little attention has been paid to the tolerance of methyl groups at cleavage subsites. In this study, the structure of GH86 β-agarase Aga86A_Wa from Wenyingzhuangia aestuarii was determined by X-ray crystallography and investigated from a structural biology perspective. The structure indicated that an accommodation pocket formed by F367, Y280, and Q326 at subsite -1 contributes to the methyl-galactose tolerance of Aga86A_Wa. Furthermore, we found that similar accommodation pockets were present in the structures of two other GH86 enzymes BuGH86 from Bacteroides uniformis and BpGH86A from Phocaeicola plebeius, and their previously undisclosed methyl-galactose tolerance was verified, validating the function of the pockets. Phylogenetic analysis, structural modeling, and hydrolysis product characterization suggested that the methyl-galactose accommodation capacity at subsite -1 was prevalent in GH86 members. These findings achieve a better understanding of the function and mechanism of GH86 agaran degrading enzymes, and will facilitate the precise preparation of agaran oligosaccharides by employing defined tools.
摘要：
Agarans是从红藻中提取的硫酸化半乳聚糖，具有高度的结构复杂性，其天然甲基化通常发生在其β-d-吡喃半乳糖单元的O-6位置。虽然许多agaran降解酶，包括琼脂糖和卟啉酶，有特点，很少注意切割亚位点对甲基的耐受性。在这项研究中,通过X射线晶体学测定了文英庄产GH86β-琼脂糖酶Aga86A_Wa的结构，并从结构生物学的角度进行了研究。该结构表明，由F367，Y280和Q326在子位-1形成的容纳袋有助于Aga86A_Wa的甲基-半乳糖耐受性。此外，我们发现，类似的适应袋存在于其他两种GH86酶的结构中来自均匀拟杆菌的BuGH86和来自Phocaeicolaplebeius的BpGH86A，他们以前未公开的甲基半乳糖耐受性得到了验证，验证口袋的功能。系统发育分析,结构建模,和水解产物表征表明，在GH86成员中，亚位点-1处的甲基-半乳糖调节能力很普遍。这些发现对GH86Agaran降解酶的功能和机制有了更好的理解，并将通过使用确定的工具促进准确制备二甘醇寡糖。
