Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Copenhagen 2100, Denmark.
Department of Chemistry, University of Copenhagen, Copenhagen 2100, Denmark.
Cell Chem Biol. 2019 Feb 21;26(2):191-202.e6. doi: 10.1016/j.chembiol.2018.10.016. Epub 2018 Nov 29.
We have characterized the structure and dynamics of the carbohydrate-modifying enzyme Paenibacillus nanensis xanthan lyase (PXL) involved in the degradation of xanthan by X-ray crystallography, small-angle X-ray scattering, and hydrogen/deuterium exchange mass spectrometry. Unlike other xanthan lyases, PXL is specific for both unmodified mannose and pyruvylated mannose, which we find is correlated with structural differences in the substrate binding groove. The structure of the full-length enzyme reveals two additional C-terminal modules, one of which belongs to a new non-catalytic carbohydrate binding module family. Ca are critical for the activity and conformation of PXL, and we show that their removal by chelating agents results in localized destabilization/unfolding of particularly the C-terminal modules. We use the structure and the revealed impact of Ca coordination on conformational dynamics to guide the engineering of PXL variants with increased activity and stability in a chelating environment, thus expanding the possibilities for industrial applications of PXL.
我们通过 X 射线晶体学、小角度 X 射线散射和氘/氢交换质谱法，研究了参与降解黄原胶的碳水化合物修饰酶——巨大芽孢杆菌黄原胶裂解酶（PXL）的结构和动力学。与其他黄原胶裂解酶不同，PXL 既能特异性作用于未经修饰的甘露糖，也能特异性作用于丙酮酸化的甘露糖，我们发现这与底物结合槽中的结构差异有关。全长酶的结构揭示了两个额外的 C 末端模块，其中一个属于新的非催化碳水化合物结合模块家族。Ca2+对 PXL 的活性和构象至关重要，我们表明螯合剂去除 Ca2+会导致 C 末端模块的局部失稳/去折叠。我们利用结构和揭示的 Ca2+配位对构象动力学的影响，指导 PXL 变体的工程设计，以提高其在螯合环境中的活性和稳定性，从而扩大 PXL 在工业应用中的可能性。
