Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, 2001 Universidad Av., Cuernavaca, Morelos 62210, Mexico; Instituto de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos, 1001 Universidad Av., Cuernavaca, Morelos 62209, Mexico.
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.
J Struct Biol. 2021 Jun;213(2):107740. doi: 10.1016/j.jsb.2021.107740. Epub 2021 May 5.
The multi-copper oxidase from the hyper-thermophilic bacteria Thermus thermophilus (Tth-MCO), has been previously characterized and described as an example of a laccase with low catalytic properties, especially when it is compared with the activity of fungal laccases, but it is active at high temperatures. Structurally, Tth-MCO has a unique feature: a β-hairpin near the T1Cu site, which is not present in any other laccases deposited at the PDB. This β-hairpin has an expected crystallographic behavior in solvent-exposed areas of a crystallized protein: lack of electron density, high B-values and several crystalline contacts with neighboring crystallographic copies; however, its dynamical behavior in solution and its biological implications have not been described. Here, we describe four new Tth-MCO crystallographic structures, and the β-hairpin behavior has been analyzed by molecular dynamics simulations, considering the effect of pH and temperature. The β-hairpin new crystallographic conformations described here, together with their dynamics, were used to understand the pH-restrained laccase activity of Tth-MCO against substrates as syringaldazine. Remarkably, there are insertions in laccases from Thermus and Meiothermus genus, sharing the same position and a methionine-rich composition of the Tth-MCO β-hairpin. This unique high methionine content of the Tth-MCO β-hairpin is responsible to coordinate, Ag and Hg in oxidative conditions, but Cu and Cu are not coordinated in crystallographic experiments, regardless of the redox conditions; however, Ag addition does not affect Tth-MCO laccase activity against syringaldazine. Here, we propose that the pH-dependent β-hairpin dynamical behavior could explain, at least in part, the inefficient laccase activity displayed by Tth-MCO in acidic pH values.
来自嗜热细菌 Thermus thermophilus（Tth-MCO）的多铜氧化酶以前被表征和描述为一种催化性能较低的漆酶的例子，特别是与真菌漆酶的活性相比，但它在高温下具有活性。结构上，Tth-MCO 具有一个独特的特征：T1Cu 位点附近的β发夹，在任何其他沉积在 PDB 的漆酶中都不存在。这种β发夹在结晶蛋白的溶剂暴露区域具有预期的晶体学行为：缺乏电子密度、高 B 值和与相邻晶体拷贝的几个晶体接触；然而，其在溶液中的动力学行为及其生物学意义尚未被描述。在这里，我们描述了四个新的 Tth-MCO 晶体结构，通过分子动力学模拟分析了β发夹的行为，考虑了 pH 值和温度的影响。这里描述的β发夹新的晶体构象及其动力学，用于理解 Tth-MCO 对作为邻苯二胺的底物的 pH 限制漆酶活性。值得注意的是，Thermus 和 Meiothermus 属的漆酶中有插入，它们共享相同的位置和 Tth-MCOβ发夹的富含蛋氨酸的组成。Tth-MCOβ发夹的这种独特的高蛋氨酸含量负责在氧化条件下配位 Ag 和 Hg，但在晶体学实验中，Cu 和 Cu 都没有配位，无论氧化还原条件如何；然而，Ag 的添加不会影响 Tth-MCO 漆酶对邻苯二胺的活性。在这里，我们提出 pH 依赖性β发夹动力学行为至少可以部分解释 Tth-MCO 在酸性 pH 值下显示的低效漆酶活性。
