NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions
NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions
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DOI：
10.1073/pnas.1208822109
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发表时间：
2012-11-13
影响因子：
11.1
通讯作者：
Vaaje-Kolstad, Gustav
中科院分区：
文献类型：
--
作者：
Aachmann, Finn L.;Sorlie, Morten;Vaaje-Kolstad, Gustav
目前被分类为碳水化合物结合模块家族33 （CBM33）和糖苷水解酶家族61 （GH61）的多糖单加氧酶可能在未来的生物精制中发挥重要作用。然而，其前所未有的催化活性的分子基础在很大程度上仍然未知。我们已经使用核磁共振技术和等温滴定量热法来解决CBP21的结构和功能方面，一个几丁质活性CBM33。核磁共振结构和弛豫研究表明，CBP21是一个致密的刚性分子，唯一的例外是催化金属结合位点。核磁共振数据进一步表明，催化中心的His28和His114结合多种二价金属离子，对Cu2+有明显的偏好（K-d = 55 nM；来自等温滴定量热法），对Cu1+有较高的偏好（K-d类似于1 nM；来自实验测定的CBP21-Cu2+的275 mV的热力学循环氧化还原电位）。Cu1+的强结合也反映在组氨酸的pK(a)值分别降低了3.6和2.2 pH单位。氰化物，一种分子氧的模拟物，被发现只与金属离子结合。这些数据支持一个模型，即铜在酶上通过外部提供的电子被还原，然后是氧结合和内部电子转移的激活。在H-2/H-1交换实验中，CBP21与晶体底物的相互作用被映射，表明底物结合涉及一个扩展的平面结合面，包括金属结合位点。这种平面催化表面似乎很适合与晶体底物相互作用。
Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu2+ (K-d = 55 nM; from isothermal titration calorimetry) and higher preference for Cu1+ (K-d similar to 1 nM; from the experimentally determined redox potential for CBP21-Cu2+ of 275 mV using a thermodynamic cycle). Strong binding of Cu1+ was also reflected in a reduction in the pK(a) values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a H-2/H-1 exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.