Romero Jefferson O, Fernández-Fueyo Elena, Avila-Salas Fabián, Recabarren Rodrigo, Alzate-Morales Jans, Martínez Angel T
Centro de Bioinformática, Simulacion y Modelado (CBSM), Departamento de Bioinformática, Facultad de Ingeniería, Universidad de Talca, 2 Norte 685, Casilla 721, Talca, Chile.
Doctorado en Ciencias Mencion Investigacion y Desarrollo de Productos Bioactivos, Instituto de Química de Recursos Naturales, Universidad de Talca, 2 Norte 685, Casilla 747, Talca, Chile.
Comput Struct Biotechnol J. 2019 Jul 10;17:1066-1074. doi: 10.1016/j.csbj.2019.07.002. eCollection 2019.
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 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.
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过程中起着关键作用。了解这种酶与其底物相互作用的分子决定因素对于合理设计用于生物技术应用的工程化过氧化物酶至关重要。在此，我们结合计算和实验方法来分析LiP（同工酶H8）与VA及其自由基阳离子（VA，由该酶氧化底物产生）之间的相互作用。在半经验量子力学水平（SQM）上计算LiP与VA/VA之间的相互作用能，能够确定在催化性色氨酸171酸性环境中参与主要相互作用的那些残基。然后，通过定向诱变产生了一系列在这些残基（谷氨酸168、天冬氨酸165、谷氨酸250、天冬氨酸264和苯丙氨酸267）处有单突变和多突变的变体，并测定了它们在VA和另外两种底物上的动力学参数。实验结果表明，谷氨酸168和谷氨酸250对于VA的结合至关重要，谷氨酸250也有助于酶的周转。通过对VA/VA与具有每个单突变的LiP之间相互作用能的新计算，进一步解释了实验结果。最后，用量子力学/分子力学计算（QM/MM）确定了自旋密度的离域，进一步支持了谷氨酸250对色氨酸171处VA氧化的贡献。
