Multi-discipline Center, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Sci Rep. 2018 Sep 24;8(1):14252. doi: 10.1038/s41598-018-32446-7.
The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli. The laccase activity of CueO G304K mutant is higher than wild-type CueO. To explain this increase in activity, we solved the crystal structure of G304K mutant at 1.49 Å. Compared with wild-type CueO, the G304K mutant showed dramatic conformational changes in methionine-rich helix and the relative regulatory loop (R-loop). We further solved the structure of Cu-soaked enzyme, and found that the addition of Cu ions induced further conformational changes in the R-loop and methionine-rich helix as a result of the new Cu-binding sites on the enzyme's surface. We propose a mechanism for the enhanced laccase activity of the G304K mutant, where movements of the R-loop combined with the changes of the methionine-rich region uncover the T1 Cu site allowing greater access of the substrate. Two of the G304K double mutants showed the enhanced or decreased laccase activity, providing further evidence for the interaction between the R-loop and the methionine-rich region. The cuprous oxidase activity of these mutants was about 20% that of wild-type CueO. These structural features of the G304K mutant provide clues for designing specific substrate-binding mutants in the biotechnological applications.
多铜氧化酶 CueO 参与大肠杆菌的铜稳态和铜（Cu）耐受。CueO G304K 突变体的漆酶活性高于野生型 CueO。为了解释这种活性的增加，我们解析了 G304K 突变体在 1.49Å分辨率下的晶体结构。与野生型 CueO 相比，G304K 突变体在富含蛋氨酸的螺旋和相对调节环（R 环）中表现出明显的构象变化。我们进一步解析了 Cu 浸泡酶的结构，发现由于酶表面上新的 Cu 结合位点，Cu 离子的加入诱导 R 环和富含蛋氨酸的螺旋进一步构象变化。我们提出了 G304K 突变体增强漆酶活性的机制，其中 R 环的运动与富含蛋氨酸区域的变化相结合，暴露出 T1 Cu 位点，从而使底物更容易进入。两个 G304K 双突变体表现出增强或降低的漆酶活性，为 R 环和富含蛋氨酸区域之间的相互作用提供了进一步的证据。这些突变体的铜氧化酶活性约为野生型 CueO 的 20%。这些 G304K 突变体的结构特征为设计生物技术应用中特定的底物结合突变体提供了线索。
