⁵¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.
⁵¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.
复制标题
钒氯过氧化物酶的 V NMR 晶体学及其定向进化 P395D/L241V/T343A 突变体：活性位点的质子化环境。
DOI：
10.1021/jacs.5b02635
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发表时间：
2015
影响因子：
15
通讯作者：
Polenova,Tatyana
中科院分区：
文献类型：
--
作者：
Gupta,Rupal;Hou,Guangjin;Renirie,Rokus;Wever,Ron;Polenova,Tatyana
钒依赖性卤代过氧化物酶（VHPOs）使用过氧化氢进行卤化物的双电子氧化。它们的机制，包括决定底物特异性和催化速率的pH依赖性的因素，知之甚少。在VHPOs的活性部位的钒酸盐辅因子含有“光谱沉默”的V（V），其在反应期间不改变氧化态。我们采用了基于51 V魔角自旋核磁共振波谱和密度泛函理论的核磁共振晶体学方法，以深入了解钒依赖性氯过氧化物酶（VCPO）在静息状态下的辅因子的结构和配位环境。在各种pH值下检查野生型VCPO及其P395 D/L241 V/T343 A突变体中的辅因子环境，其表现出5-100倍的改进的催化活性。由于快速MAS探针技术获得的最佳灵敏度使分配的位置和数量的质子上的钒酸盐作为pH值的函数。钒酸盐辅因子改变其质子化从四重质子化在pH 6.3三重质子化在pH 7.3双质子化在pH 8.3。相反，在突变体中，钒酸质子化是相同的pH 5.0和8.3，和辅因子是双质子化。这种方法来确定不同的质子化环境的辅因子，这也是pH值依赖性，可以帮助解释不同的反应性的野生型和突变体VCPO和它们的pH值依赖性。这项研究表明，51 V NMR晶体学可以用来推导钒中心在大生物分子中的详细配位环境。
Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains “spectroscopically silent” V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on51V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5–100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that51V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.