1,2-β-Mannobiose phosphorylases (1,2-β-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-β-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-β-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-β-MBPs and should serve as an important guidance for enzyme engineering for further applications.

来自糖苷水解酶 130 (GH130) 家族的 1,2-β-甘露二糖磷酸化酶 (1,2-β-MBP) 是糖化学应用中重要的生物催化剂，因为它们具有合成寡甘露聚糖的能力。在这里，我们报告了来自热厌氧菌的耐热 1,2-β-MBP 的晶体结构sp. X-514 命名为 Teth514_1789 以揭示其更高的热稳定性和作用机制的分子基础。我们还解析了甘露糖、1-磷酸甘露糖 (M1P) 和 1,4-β-甘露二糖的酶复合物，以展示三个主要亚位点的酶-底物相互作用网络。值得注意的是，在活性位点发现了一个应该来自结晶缓冲液的 Zn 离子，它与 M1P 的磷酸盐部分相协调。尽管如此，这种锌配位应该反映抑制状态，因为补充锌会严重损害酶活性。这些结果表明在应用Teth514_1789和其他相关酶时应考虑金属离子的影响。基于该结构，共享 61 的 Teth514_1788 的可靠模型。与 Teth514_1789 有 7% 的序列同一性，但显示出不同的底物偏好。分析这两种密切相关酶的结构特征，我们假设覆盖催化中心入口的环片段的长度可能会调节底物选择性。总之，这些信息提供了对 GH130 1,2-β-MBP 的深入理解，并应作为酶工程进一步应用的重要指导。