期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 3, 页码 1504-1513出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1904889117
关键词
LPMO; peroxygenase; biomass; chitin; QM/MM
资金
- Research Council of Norway [240967, 269408]
- National Science Foundation [MCB1715176, ACI-1548562, TG-MCB090159]
- US Department of Energy (DOE) [DE-AC36-08GO28308]
- US DOE Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office
- Saga Cluster [NN1003K]
- Abel Cluster [NN1003K]
Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O-2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O-2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.
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