Seonah Kim^a, Jerry Stahlberg^b,c, Mats Sandgren^b, Robert S. Paton^d,1, and Gregg T. Beckham^a,1

^aNational Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80202;
^bDepartment of Molecular Biology, Swedish University of Agricultural Sciences, SE 75007 Uppsala, Sweden;
^cDepartment of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, NO-1432 As, Norway; and
^dChemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, United Kingdom

Abstract
Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η¹-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η¹) to copper, and that a copper-oxyl-mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.

C-H activation, copper monooxygenase, GH61, CBM33, biofuels

¹To whom correspondence may be addressed.

Author contributions: S.K. performed research; S.K., J.S., M.S., R.S.P., and G.T.B. analyzed data; and S.K., J.S., M.S., R.S.P., and G.T.B. wrote the paper.