Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from Serratia marcescens SmAA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme–substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H₂O, O₂, and H₂O₂ can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of SmAA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies.

中文翻译：

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溶多糖单加氧酶如何结合结晶几丁质

溶菌多糖单加氧酶（LPMO）在自然界和生物炼制业中都是生物质转化的主要参与者。如何将单铜LPMO活性位点相对于晶体基质表面定位以催化强大的，但具有潜在自毁性的氧化化学反应是该领域的主要问题之一。我们采用了多学科的方法，结合了生化，光谱和分子建模方法，通过对粘质沙雷氏菌（Serratia marcescens Sm）的精心研究的LPMO研究甲壳质的结合。AA10A（或CBP21）。通过分析酶切割模式确定酶在单链底物上的取向。在此分析的基础上，进行了分子动力学（MD）模拟，以研究LPMO与结晶甲壳质的三种不同表面拓扑之间的相互作用。最终的原子模型表明，大多数酶与底物的相互作用都涉及要被切割的多糖链。这些模型还揭示了受约束的活性位点几何形状以及将主体溶剂连接到铜位点的通道，只有小分子，例如H ₂ O，O ₂和H ₂ O ₂才能通过该通道通过可以扩散。此外，MD模拟，量子力学/分子力学计算和电子顺磁共振波谱表明，与底物结合时，Cu配位水分子的重排是必要的，并且还为实验观察到的Sm AA10A的C1氧化区域特异性提供了理论依据。这项研究为LPMO与结晶几丁质之间的相互作用提供了第一个受实验支持的原子观。催化中心的限制对于控制LPMO进行的氧化化学反应至关重要，这将有助于指导未来的机理研究。