Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using "Peroxidase-like" Myoglobin Mutants and "Myoglobin-like" DHP Mutants
Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using "Peroxidase-like" Myoglobin Mutants and "Myoglobin-like" DHP Mutants
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DOI：
10.1021/bi2009129
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发表时间：
2011-09-27
期刊：
影响因子：
2.9
通讯作者：
Dawson, John H.
中科院分区：
文献类型：
--
作者：
Du, Jing;Huang, Xiao;Dawson, John H.
脱卤过氧化物酶 (DHP) 在海洋多毛类动物 Amphitrite ornata 中发现，是第一个具有过氧化物酶活性的含血红素球蛋白。 DHP 的序列和晶体结构表明它是从古老的 O-2 运输和存储球蛋白进化而来的。因此，DHP保留了氧载体功能，但也具有降解其生活环境中的卤代酚毒物的能力。抹香鲸肌红蛋白 (Mb) 在三价铁状态下具有过氧化物酶活性，比 DHP 低 10 倍。在 DHP 中观察到的催化活性增强似乎主要是由于 DHP 进化过程中出现的近端和远端组氨酸残基位置的细微变化而产生的。在此，我们报告了通过检查“过氧化物酶样”Mb 突变体和“Mb 样”DHP 突变体而得出的 DHP 作用机制的研究。野生型 Mb 的脱卤能力在过氧化物酶样 Mb 突变体（F43H/H64L、G6ST 和 G65I Mb）中增强，但在 Mb 样 T56G DHP 变体中减弱。 X射线晶体学数据显示，G65T Mb和G65I中的远端His残基的位置分别类似于0.3埃和类似于0.8埃，与野生型蛋白相比，距离血红素铁更远。 H93K/T95H 双突变体 Mb（近端 His 转移至“DHP 样”位置）具有增加的过氧化物酶活性。此外，通过在His89附近引入负电荷以增强近端His的咪唑酯特性，产生了更好的脱卤过氧化物酶（M86E DHP）。最后，在无外源配体的 DHP、乙酸盐结合的 DHP 和远端位点阻断剂 L100F DHP 突变体之间仅观察到脱卤活性的微小差异。因此，我们得出结论，卤代酚在内部结合位点（即远端腔）的结合对于催化不是必需的。这项工作为过氧化物酶的新结构功能范式和双功能酶 DHP 的分子进化奠定了基础。
Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O-2 transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity, similar to 10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G6ST, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned, similar to 0.3 and similar to 0.8 angstrom, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.