Breslmayr Erik, Poliak Peter, Požgajčić Alen, Schindler Roman, Kracher Daniel, Oostenbrink Chris, Ludwig Roland
Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria.
Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria.
Antioxidants (Basel). 2022 May 31;11(6):1096. doi: 10.3390/antiox11061096.
Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions on the activity and stability of LPMO during catalysis. Turbidimetric and photometric assays were used to identify LPMO inhibitors and measure their inhibitory effect. Selected inhibitors were employed to study LPMO activity and stability during cellulose depolymerization by HPLC and turbidimetry. It was found that the fungal metabolic products oxalic acid and citric acid strongly reduce LPMO activity, but also protect the enzyme from deactivation. QM calculations showed that the copper atom in the catalytic site could be ligated by bi- or tridentate chelating compounds, which replace two water molecules. MD simulations and QM calculations show that the most likely inhibition pattern is the competition between the inhibitor and reducing agent in the oxidized Cu(II) state. A correlation between the complexation energy and the IC values demonstrates that small, bidentate molecules interact strongest with the catalytic site copper and could be used by the fungus as physiological effectors to regulate LPMO activity.
裂解多糖单加氧酶（LPMOs）广泛分布于真菌中，催化纤维素等多糖的氧化降解。尽管其名称如此，但LPMOs具有显著的过氧化物酶活性，这体现在高周转数上，但也会导致失活。我们报告了小分子和离子在催化过程中对LPMO活性和稳定性的影响。采用比浊法和光度法鉴定LPMO抑制剂并测量其抑制效果。选用抑制剂通过高效液相色谱法和比浊法研究纤维素解聚过程中LPMO的活性和稳定性。结果发现，真菌代谢产物草酸和柠檬酸能强烈降低LPMO活性，但也能保护该酶不被失活。量子力学计算表明，催化位点中的铜原子可被双齿或三齿螯合化合物配位，这些化合物取代了两个水分子。分子动力学模拟和量子力学计算表明，最可能的抑制模式是抑制剂与氧化态Cu（II）中的还原剂之间的竞争。络合能与IC值之间的相关性表明，小的双齿分子与催化位点铜的相互作用最强，真菌可将其用作生理效应物来调节LPMO活性。
