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首页> 外文期刊>Biochemistry >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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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

机译：孟菲斯火山口脱卤素过氧化物酶（DHP）：影响使用“过氧化物酶样”肌红蛋白突变体和“肌红蛋白样” DHP突变体影响卤代苯酚脱卤机理的结构因素的研究

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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.
机译：脱卤过氧化物酶（DHP）是在海洋类多头类多头动物Amphitrite ornata中发现的，是第一种具有血红素的过氧化物酶活性球蛋白。 DHP的序列和晶体结构认为，它是从古老的O（2）运输和存储球蛋白演变而来的。因此，DHP保留了氧的载流子功能，但还具有在其生活环境中降解卤代苯酚有毒物质的能力。铁状态的抹香鲸肌红蛋白（Mb）具有过氧化物酶活性，大约是DHP的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。本文中，我们报告了对DHP作用机理的研究，该作用源于“过氧化物酶样” Mb突变体和“ Mb类” DHP突变体的检查。在过氧化物酶样的Mb突变体（F43H / H64L，G6ST和G65I Mb）中，野生型Mb的脱卤能力增强，而在Mb样T56G DHP变体中减弱。 X射线晶体学数据显示，与野生型蛋白相比，G65T Mb和G65I的远端His残基的位置与血红素铁分别更接近0.3和0.8埃。 H93K / T95H双突变体Mb的近端His移至“ DHP样”位置，具有增加的过氧化物酶活性。此外，通过在His89附近引入负电荷来增强近端His的咪唑化物特征，可以产生更好的脱卤过氧化物酶（M86E DHP）。最后，在无外源配体的DHP，结合乙酸盐的DHP和远端位阻滞剂L100F DHP突变体之间，仅观察到最小的脱卤活性差异。因此，我们得出结论，卤代酚在内部结合位点（即远端腔）中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的分子进化提供了基础。

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