The catalytic properties of Thermus thermophilus SG0.5JP17-16 laccase were regulated by the conformational dynamics of pocket loop 6
The catalytic properties of Thermus thermophilus SG0.5JP17-16 laccase were regulated by the conformational dynamics of pocket loop 6
复制标题
嗜热栖热菌SG0.5JP17-16漆酶的催化特性受口袋环6的构象动力学调节
DOI：
10.1016/j.bbagen.2021.129872
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发表时间：
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通讯作者：
Xiaorong Yang
中科院分区：
文献类型：
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作者：
Yi Zhang;Zhuojun Dai;Shumin Zhang;Xiaorong Yang
背景漆酶是蓝色多铜氧化酶家族的成员之一。它可以催化各种底物的氧化。嗜热嗜热菌SG0.5JP17-16漆酶(LacTT)是一种耐热、耐pH、耐卤化物的漆酶，对合成染料有一定的脱色作用。方法利用位于环6中的Asp394和Asp396残基，研究环6对漆酶催化性能的影响。对两种氨基酸进行了定点突变。采用动力学方法对突变体的催化效率进行了表征。利用具有不同催化活性的突变体对合成染料进行脱色实验，以明确催化效率与染料脱色的关系。结果漆酶活性较低的D394M、D394E和D394R突变体漆酶脱色效率降低，而催化效率较高的D396A、D396M和D396E突变体酶对合成染料的脱色效率高于野生型酶。D394残基通过底物通道入口处含有D396残基的氨基酸相互作用网络控制这种构象变化。这些研究可能为提高漆酶活性以更好地应用于工业应用提供线索，和/或有助于进一步了解漆酶在底物上的氧化机理。
BackgroundLaccase is one member of the blue multicopper oxidase family. It can catalyze the oxidation of various substrates. TheThermus thermophilusSG0.5JP17-16 laccase (lacTT) is thermostable, pH-stable, and high tolerance to halides, and can decolorize the synthetic dyes. In lacTT, the function of the loop 6 constructing the substrate-binding pocket wasn't clear.MethodsThe residues Asp394 and Asp396 located in loop 6, and were used to probe how the loop 6 influenced catalytic properties of the laccase. Site-directed mutagenesis was performed for two amino acids. Kinetic assay was utilized to characterize the catalytic efficiency of mutants. Mutants with different catalytic activities were used to decolorize the synthetic dyes to clarify the relationship between the catalytic efficiency and dye decolorization. Redox potential, structural and spectral analyses were performed to explain the differences in laccase activity between wild type and mutant enzymes.ResultsD394M, D394E and D394R mutants with the lower laccase activity displayed a decreased decolorization efficiency, while D396A, D396M and D396E mutant enzymes with higher catalytic efficiency decolorized the synthetic dye more efficiently than the wild type enzyme.ConclusionsThe pocket loop 6 might experience a conformational dynamics. The D394 residue controlled this conformation change by amino acid interaction networks containing the D396 residue at the entrance of substrate channel.General significancesThese studies may provide clues to improve the activity of the laccase for the better use in industrial applications, and/or contribute to further understanding the mechanism of laccase oxidation on the substrate.