Institute of Microbiology of the CAS, Division BioCeV, Prumyslova 595, 252 50 Vestec, Czech Republic.
Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 43 Prague 2, Czech Republic.
Biomolecules. 2020 Feb 5;10(2):242. doi: 10.3390/biom10020242.
Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in LPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of LPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.
溶细胞多糖单加氧酶（LPMOs）是在木质纤维素糖化中应用的具有工业重要性的氧化还原酶。我们使用先进的时间分辨质谱技术，阐明了真菌 LPMO9C 中与底物介导的催化稳定性相关的结构决定因素。LPMO 需要还原活性部位的铜才能具有催化活性。我们表明，LPMO9C 中的铜还原会导致活性部位周围的结构重排和紧凑化。然而，更长时间暴露于还原剂抗坏血酸也会引发结合氧物种的解偶联反应，导致氧化损伤、部分展开，甚至 LPMO9C 的片段化。有趣的是，在氧化或还原的 LPMO 与结晶纤维素孵育时，没有检测到氢/氘交换率的变化，这表明 LPMO-底物相互作用主要是侧链介导的，既不影响蛋白质内部氢键，也不会引起蛋白质表面的显著屏蔽。另一方面，我们观察到底物的保护作用，它减缓了解偶联反应引起的自动氧化损伤。这些观察结果进一步补充了 LPMO 催化过程中结构变化的情况。
