Paramagnetic nuclear magnetic resonance relaxation and molecular mechanics studies of the chloroperoxidase-indole complex: insights into the mechanism of chloroperoxidase-catalyzed regioselective oxidation of indole.
Paramagnetic nuclear magnetic resonance relaxation and molecular mechanics studies of the chloroperoxidase-indole complex: insights into the mechanism of chloroperoxidase-catalyzed regioselective oxidation of indole.
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DOI：
10.1021/bi4002437
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发表时间：
2013-05-28
期刊：
影响因子：
2.9
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
Zhang R;He Q;Chatfield D;Wang X
为了揭示cpo催化吲哚区域选择性氧化的机理，利用核磁共振弛豫测量和计算技术研究了cpo -吲哚配合物的结构。cpo -吲哚配合物的解离常数（KD）约为21 mM。通过核磁共振弛豫测量和分子对接计算得到的吲哚与血红素铁之间的质子距离(r)表明，吲哚的吡咯环指向血红素，其2-H直接指向血红素铁。KD和r值在3.0 ~ 6.5范围内与pH无关。cpo -吲哚配合物的稳定性和结构也与氯离子/碘离子浓度无关。分子对接表明，在吲哚与CPO结合过程中，吲哚的N-H与Glu 183的羧基O之间形成了一个氢键。利用核磁共振r值作为距离约束对CPO-吲哚配合物进行模拟退火，结果表明，吲哚与CPO结合时的范德华相互作用比库仑相互作用强得多，表明吲哚与CPO的结合主要受疏水相互作用而非静电相互作用支配。这项工作为长期寻求的机制提供了第一个实验和理论证据，该机制导致cpo催化吲哚氧化的“意想不到的”区域选择性。CPO-吲哚配合物的结构将成为指导CPO突变体设计的灯塔，为生物技术应用量身定制活性。
To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the structure of the CPO-indole complex was studied using NMR relaxation measurements and computational techniques. The dissociation constant (KD) of the CPO-indole complex was calculated to be approximately 21 mM. The distances (r) between protons of indole and the heme iron calculated from NMR relaxation measurements and molecular docking revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. Both KD and r values are independent of pH in the range of 3.0–6.5. The stability and structure of the CPO-indole complex are also independent of the concentration of chloride/iodide ion. Molecular docking suggests the formation of a hydrogen bond between the N–H of indole and the carboxyl O of Glu 183 in the binding of indole to CPO. Simulated annealing of the CPO-indole complex using r values from NMR experiments as distance restraints reveals that the van der Waals interactions were much stronger than the Coulomb interactions in indole binding to CPO, indicating that the association of indole with CPO is primarily governed by hydrophobic rather than electrostatic interactions. This work provides the first experimental and theoretical evidence for the long-sought mechanism that leads to the “unexpected” regioselectivity of CPO-catalyzed oxidation of indole. The structure of the CPO-indole complex will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications.
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