Department of Chemistry and Stanford ChEM-H, Stanford University, Stanford, California, USA.
Stanford ChEM-H Macromolecular Structure Knowledge Center, Stanford University, Stanford, California, USA.
J Biol Chem. 2022 May;298(5):101917. doi: 10.1016/j.jbc.2022.101917. Epub 2022 Apr 9.
Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often associated with positive health outcomes. The abundance of A. muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although A. muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymatic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc_0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 Å resolution) revealed O-glycan-binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using molecular modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition.
阿克曼氏菌（Akkermansia muciniphila）是一种存在于人类肠道中的黏液降解微生物，通常与健康的结果呈正相关。A. muciniphila 的丰度受到营养素的存在和可及性的调节，这些营养素可以来自饮食或宿主糖蛋白。特别是，降解宿主黏液素的能力，即一类携带密集 O-糖基化结构域的蛋白质，为其在持续定植黏膜生态位方面提供了竞争优势。尽管已知 A. muciniphila 依赖黏液素作为碳源和氮源，但该微生物在肠道中处理黏液素的酶机制尚未完全阐明。在这里，我们重点关注黏液素选择性金属蛋白酶 Amuc_0627（AM0627），它已知能在携带截断核心 1 O-聚糖的相邻残基之间切割。我们表明，这种酶能够降解纯化的黏液素 2（MUC2），MUC2 是肠道黏液的主要蛋白质成分。AM0627 的 X 射线晶体结构（1.9 Å 分辨率）揭示了 O-聚糖结合残基，这些残基在同一家族中具有结构特征的酶之间是保守的。我们进一步通过分子建模来合理化底物切割基序，以识别非保守的聚糖相互作用残基。我们得出结论，这些残基的突变导致了对底物偏好的改变，甚至达到了聚糖水平，这为 O-聚糖识别的结构决定因素提供了深入的了解。
