Abstract
ABSTRACTLytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes, broadly distributed across the Tree of Life. We recently reported that LPMOs can use H2O2 as an oxidant, revealing a novel reaction pathway. Here, we aimed to elucidate the H2O2-mediated reaction mechanism with experimental and computational approaches. In silico studies suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation, and guides the derived hydroxyl radical towards formation of a copper-oxyl intermediate. The initial H2O2 homolytic cleavage and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are suggested to be the main energy barriers. Under single turnover conditions, stopped-flow fluorimetry demonstrates that LPMO-Cu(II) reduction to Cu(I) is a fast process compared to the re-oxidation reactions. We found that re-oxidation of LPMO-Cu(I) is 2000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. In agreement with the notion of ternary complex formation, when chitin is added, re-oxidation by H2O2 is accelerated whereas that by O2 slows. Simulations indicated that Glu60, a highly-conserved residue, gates the access to the confined active site and constrains H2O2 during catalysis, and Glu60 mutations significantly decreased the enzyme performance. By providing molecular and kinetic insights into the peroxygenase activity of chitinolytic LPMOs, this study will aid the development of applications of enzymatic and synthetic copper catalysis and contribute to understanding pathogenesis, notably chitinolytic plant defenses against fungi and insects.
Publisher
Cold Spring Harbor Laboratory
Cited by
2 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献