Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid–base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis.

中文翻译：

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口腔病原体福赛坦纳菌的稳定、广泛特异性多聚唾液酸酶的结构和功能表征


唾液酸酶是针对末端唾液酸和底层糖之间的糖苷键的糖基水解酶。连翘坦纳菌 (Tannerella forsythia) 的 NanH 唾液酸酶是与严重牙周病相关的细菌之一，在毒力中发挥着重要作用。在这里，我们证明这种广泛特异性的酶（但对 α2,3 的亲和力高于 α2,6 连接的唾液酸）可以消化复杂的聚糖，但不能消化含有 Neu5,9Ac 的聚糖。此外，我们证明它是一种高度稳定的二聚酶，并对脱辅基形式的天然酶以及与唾液酸类似物/抑制剂（奥司他韦）的复合物进行了彻底的结构分析。我们还使用非催化 (D237A) 变体来表征与天然底物 3- 和 6-唾液酸乳糖复合时的分子相互作用。该数据集还揭示了 NanH 碳水化合物结合模块（CBM、CAZy CBM 93）具有由反平行 β 链组成的新颖折叠。催化结构域结构包含新颖的特征，包括非脯氨酰顺式肽和靠近活性位点的不常见的精氨酸侧链旋转异构体（R306）。通过诱变程序，我们鉴定了关键活性位点残基（D237、R212 和 Y518），并探讨了 2,3 和 2,6 连接底物内糖苷键附近残基突变的影响。这些数据表明，R306 以及与酸碱催化剂 D237 相邻的残基 S235 和 V236 的诱变影响了这种细菌唾液酸酶的连接特异性偏好，为糖技术应用的酶工程开辟了可能性，并为计算机设计提供了关键的结构信息。该酶的特异性抑制剂用于治疗牙周炎。