Neutrons Identify Oxygen Activation in LPMOs

Fungal lytic polysaccharide monooxygenases (LPMOs)
Scientists used neutrons to probe the structural details of the specialized LPMO fungal enzyme that relies on oxidation to digest molecules, aiming to improve the efficiency of enzymatic cellulose breakdown. [From O’Dell, W. B., P. K. Agarwal, and F. Meilleur. “Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.” Angew. Chem. Int. Ed. 56, 767–770 (2017). [DOI:10.1002/anie.201610502]. ©2017 The Authors. Published by Wiley-VCH Verla GmbH & Co. KGaA, Weinheim.]
Fungal lytic polysaccharide monooxygenases (LPMOs) are known to enhance the efficiency of cellulose-hydrolyzing enzymes through oxidative cleavage of the glycosidic bonds. For this study, PMO-2 from Neurospora crassa was heterologously expressed from Pichia pastoris, purified, and crystallized for high-resolution X-ray crystal structures that revealed “prebound” molecular oxygen in the resting state and a dioxo species in complex with the catalytic copper (Cu2+) ion, which is the first structural description of molecular oxygen (O2) activation by a LPMO. In addition, neutron diffraction studies and density functional theory calculations have identified a role for a conserved histidine in promoting oxygen activation. Extension of these studies to the enzyme-substrate complex could provide a complete picture of the enzymatic mechanism for the potential benefit of applications such as bioethanol production.

O’Dell, W. B., et al. “Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.” Angew. Chem. Int. Ed. 129(3), 785–788 (2017). [DOI:10.1002/anie.201610502].

Instruments and Facilities Used: Neutron crystallography. Joint X-ray/neutron refinement at Center for Structural Molecular Biology at Oak Ridge National Laboratory (ORNL). Diffraction data were collected at SER-CAT 22-ID at the Advanced Photon Source at Argonne National Laboratory and at CG-4D IMAGINE (NSF MRI 09229719) at the High Flux Isotope Reactor at ORNL.

Funding Acknowledgements: Protein expression and purification experiments at Center for Structural Molecular Biology (CSMB), a User Facility of the Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science. Diffraction data collected at SER‐CAT 22‐ID at Argonne National Laboratory’s (ANL) Advanced Photon Source (APS) and at CG‐4D IMAGINE (National Science Foundation [NSF] magnetic resonance imaging [MRI] 09229719) at Oak Ridge National Laboratory’s (ORNL) High Flux Isotope Reactor (HFIR), both DOE Office of Biological Energy Sciences (OBES) User Facilities. W.B.O. support: NSF IGERT 1069091. F.M. support: U.S. Department of Agriculture (USDA National Institutes of Food and Agriculture (NIFA) Hatch 211001. P.K.A. support: NIH GM105978.