Institute of Protein Research RAS, Institutskaya 4, Pushchino, Moscow 142290, Russia.
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.
Int J Mol Sci. 2019 Jun 28;20(13):3184. doi: 10.3390/ijms20133184.
Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3 and Cu3), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His165 is a "gateway" at the O-tunnel leading from solvent to the Cu3 of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His157 that belongs to the first coordination sphere of Cu3.
漆酶（EC 1.10.3.2）是一种多铜氧化还原酶，作用于二酚及其相关物质。漆酶在生物技术和环境修复中具有重要作用。这些酶含有单核 T2 铜离子和两个 T3 铜离子（Cu3 和 Cu3），形成所谓的三核中心（TNC）。除了典型的三结构域漆酶外，细菌还产生两结构域（2D）酶，它们在中性和碱性 pH 值下具有活性，热稳定且对抑制剂具有抗性。在这项工作中，我们对来自 Ac-993（SgfSL）的重组 2D 漆酶及其四个突变体形式的晶体结构和催化特性进行了比较分析，这些突变体形式替换了位于假定 T3-溶剂隧道狭窄处的两个氨基酸残基。我们获得了具有 His165、Phe 和 Ile170 取代的无活性酶，Ala 或 Phe。His165Ala 变体比野生型更活跃。我们认为 His165 是从溶剂通向酶的 Cu3 的 O-隧道的“门户”。Ile170 的侧链可能通过维持属于 Cu3 第一配位球的 His157 的咪唑环的位置间接参与 T3 中心铜离子的配位。
