Molecular mechanism of the chitinolytic peroxygenase reaction

• Published in: Proceedings of the National Academy of Sciences - PNAS 2020-01, Vol.117 (3), p.1504-1513
• Main Authors: Bissaro, Bastien, Streit, Bennett, Isaksen, Ingvild, Eijsink, Vincent G. H., Beckham, Gregg T., DuBois, Jennifer L., Røhr, Åsmund K.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocatalysis; Biological Sciences; Catalysts; Cellulose - chemistry; Cellulose - metabolism; Chitin; Chitin - chemistry; Chitin - metabolism; Computer applications; Conformation; Copper; Copper - chemistry; Copper - metabolism; Enzymes; Fluorimetry; Hydrogen bonding; Hydrogen bonds; Hydrogen peroxide; Hydrogen Peroxide - chemistry; Hydrogen Peroxide - metabolism; Hydroxyl radicals; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Organic chemistry; Oxidants; Oxidation-Reduction; Oxidizing agents; Pathogenesis; PNAS Plus; Polysaccharides; Priming; Reaction mechanisms; Reoxidation; Serratia marcescens - enzymology; Substrates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1904889117

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 O₂, 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 O₂ 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.