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首页> 美国卫生研究院文献>Computational and Structural Biotechnology Journal >Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study
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Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study

机译：木质素过氧化物酶催化藜芦醇氧化的结合及催化机理：理论和实验研究

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Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA•+, resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA•+ enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA•+ and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171.
机译：木质素过氧化物酶（LiP）及其天然底物藜芦醇（VA）在白腐真菌降解木质素中起关键作用。在合理设计工程过氧化物酶用于生物技术应用中，了解这种酶与其底物相互作用的分子决定因素至关重要。在这里，我们结合计算和实验方法来分析Phanerochaete chrysosporium LiP（同功酶H8）与VA及其自由基阳离子（VA •+ ，是由于酶对底物的氧化作用）的相互作用。 LiP和VA / VA •+ 之间在半经验量子力学水平（SQM）上进行的相互作用能计算可以识别出参与主要相互作用的催化Trp171酸性环境中的那些残基。然后，通过定向诱变产生了一系列在这些残基上具有单个和多个突变的变体（Glu168，Asp165，Glu250，Asp264和Phe267），并在VA和两个其他底物上估算了它们的动力学参数。实验结果表明，Glu168和Glu250对于VA的结合至关重要，而Glu250也有助于酶的转换。通过新计算VA / VA •+ 与LiP与每个单个突变的相互作用能进一步合理化了实验结果。最后，通过量子力学/分子力学计算（QM / MM）确定了自旋密度的离域，进一步支持了Glu250对Trp171上VA氧化的贡献。

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