PDF(Pubmed)
Abstract：
Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.
摘要：
几丁质是一种丰富的生物聚合物和病原体相关的分子模式，可刺激宿主的先天免疫反应。哺乳动物表达几丁质结合和几丁质降解蛋白以从体内去除几丁质。这些蛋白质中的一种，酸性哺乳动物几丁质酶(AMCase),是一种酶，以其在胃中的酸性条件下发挥作用的能力而闻名，但在具有更中性pH值的组织中也具有活性，比如肺。这里,我们使用了生化的组合，结构,和计算建模方法来检查小鼠同源物（mAMCase）如何在酸性和中性环境中发挥作用。我们在很宽的pH范围内测量了mAMCase活性的动力学特性，在pH2和7下定量其不寻常的双重活性最佳。我们还解决了与寡聚GlcNAcn复合的mAMCase的高分辨率晶体结构，甲壳素的积木，在那里我们确定了广泛的构象配体异质性。利用这些数据，我们进行了分子动力学模拟，表明在两个环境pH范围内，关键催化残基如何通过不同的机制质子化。这些结果结合了结构，生物化学，和计算方法，以提供对不同pH下控制mAMCase活性的催化机理的更完整的理解。具有可调pH优化的工程蛋白质可能为开发改进的酶变体提供新的机会，包括AMCase,用于甲壳素降解的治疗目的。
