Molecular mechanism of the chitinolytic peroxygenase reaction
Molecular mechanism of the chitinolytic peroxygenase reaction
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DOI：
10.1073/pnas.1904889117
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发表时间：
2020-01-21
影响因子：
11.1
通讯作者：
Rohr, Asmund K.
中科院分区：
文献类型：
--
作者：
Bissaro, Bastien;Streit, Bennett;Rohr, Asmund K.
裂解多糖单加氧酶（LPMO）是最近发现的一类广泛分布于生命树中的单铜酶。最近的报道表明，LPMO可以使用H2 O2作为氧化剂，从而进行一种新型的过氧合酶反应，涉及前所未有的铜化学。在这里，我们提出了一个相结合的计算和实验分析过氧化氢介导的反应机制。在硅片的研究，基于一个模型的酶在复杂的晶体基板，表明氢键网络，涉及酶和基板，使过氧化氢到应变反应构象和指导衍生的羟基自由基朝向形成的铜氧基中间体。H_2O_2的初始裂解和随后的铜氧中间体从甲壳素中夺取氢原子是主要的能垒。停流荧光实验表明，与再氧化反应相比，LPMO-Cu（II）到LPMO-Cu（I）的引发还原是一个快速过程。使用导致单一氧化事件的条件，我们发现LPMO-Cu（I）的再氧化用H2 O2比用O-2快2，000倍，后者比其它单加氧酶报道的速率慢几个数量级。底物的存在加速了H2 O2的再氧化，而O-2的再氧化变得较慢，支持过氧合酶的范例。这些见解的过氧合酶性质的LPMOs将有助于开发和应用的酶和合成铜催化剂，并有助于进一步了解LPMOs在自然界中的作用，从生物质转化到几丁质分解的发病机制。
Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O-2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O-2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.