Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
Department of Biomedicine, University of Bergen, Bergen, Norway.
Proteins. 2020 May;88(5):654-668. doi: 10.1002/prot.25846. Epub 2019 Nov 21.
Human pathogenic and commensal bacteria have evolved the ability to scavenge host-derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known-here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N-acetyl-d-glucosamine, N-acetyl-d-galactosamine and N-acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N-acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate-a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism.
人体病原和共生细菌已经进化出了从宿主中获取唾液酸并将其降解为营养物质的能力。大肠杆菌 yjhBC 操纵子的表达受 repressor 蛋白 nanR 控制，该蛋白调节负责唾液酸摄取和代谢加工的核心机器。yjhBC 编码蛋白的作用尚不清楚——在这里，我们证明 yjhC 编码的酶是一种参与细菌唾液酸降解的氧化还原酶/脱氢酶。首先，我们通过敲除实验证明 yjhC 广泛参与碳水化合物代谢，包括 N-乙酰-d-葡萄糖胺、N-乙酰-d-半乳糖胺和 N-乙酰神经氨酸。差示扫描荧光法表明 yjhC 结合 N-乙酰神经氨酸及其内酯变体以及 NAD(H)，这与其氧化还原酶的作用一致。接下来，我们以 1.35 Å 的分辨率解决了 yjhC 与 NAD(H)辅因子复合物的晶体结构。该蛋白折叠属于 Gfo/Idh/MocA 蛋白家族。在晶体形式中观察到的二聚体组装通过溶液研究得到证实。整体细化揭示了一个灵活的环区，该环区在催化过程中可能发挥关键作用，为底物提供必需的接触，这是 yjhC 在与其密切相关的结构中特有的特征。受结构指导，计算机对接实验支持唾液酸及其在结合口袋中的几种常见衍生物的结合，该结合口袋具有整体正电荷分布。总之，我们的结果验证了 yjhC 作为一种真正的氧化还原酶/脱氢酶的作用，并提供了支持其参与唾液酸代谢的第一个证据。
