School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei, Anhui, China; Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institute, Anhui University, 111 Jiulong Road, Hefei, Anhui, China.
School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei, Anhui, China.
Biochem Biophys Res Commun. 2019 Sep 10;517(1):29-35. doi: 10.1016/j.bbrc.2019.06.150. Epub 2019 Jul 23.
In this paper, we characterized Am2136 as a β-N-acetylhexosaminidase from Akkermansia muciniphila to perform the biochemical characteristics and the crystal structure of selenomethionine-labeled Am2136 with GlcNAc complex. Crystallographic evidence suggests that an oxazolinium ion was formed intermediately by the 2-acetamido group during the substrate-assisted catalytic procedure. Structural and kinetic analysis of native Am2136 and D412A, E413A mutants were investigated and the results revealed substantial difference. The K/K value of D412A was decreased 4297-fold compared to native Am2136 revealed that mutation of Asp-412 results in preventing the 2-acetamido substituent from providing anchimeric assistance and thus reducing the catalytic efficiency. Moreover, Am2136 has a wide dependence on pH and temperature, while sensitive to divalent metal ions such as Ca and Mn. These biochemical and crystallographic results provide evidences that Asp-412 residue assists to orient the 2-acetamido group for catalysis. Based on crystallographic evidence and sequence alignment with other GH family 20 enzymes, Asp-412 residue is possibly fundamental for Am2136 during substrate-assisted catalysis.
在本文中，我们将 Am2136 鉴定为阿克曼氏菌属黏液亚种的β-N-乙酰己糖苷酶，以研究其与 GlcNAc 复合物的生化特性和晶体结构。晶体学证据表明，在底物辅助催化过程中，2-乙酰氨基基团形成了一个恶唑啉离子中间体。我们对天然 Am2136 和 D412A、E413A 突变体进行了结构和动力学分析，结果显示出显著的差异。与天然 Am2136 相比，D412A 的 K/K 值降低了 4297 倍，表明突变天冬氨酸 412 会阻止 2-乙酰氨基取代基提供亲核辅助，从而降低催化效率。此外，Am2136 对 pH 和温度具有广泛的依赖性，但对 Ca 和 Mn 等二价金属离子敏感。这些生化和晶体学结果提供了证据，表明天冬氨酸 412 残基有助于定向 2-乙酰氨基基团进行催化。基于晶体学证据和与其他 GH 家族 20 酶的序列比对，天冬氨酸 412 残基可能是 Am2136 在底物辅助催化过程中的基础。
