title: Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering.
abstract: Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). Here, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. This work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Electronic address: annettebodenheimer@ncsu.edu.
authors.forename: Annette M
authors.initials: AM
authors.lastname: Bodenheimer
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Electronic address: wbodell@ncsu.edu.
authors.forename: William B
authors.initials: WB
authors.lastname: O'Dell
authors.affiliation: Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Electronic address: stanleycb@ornl.gov.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Stanley
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Electronic address: fmeille@ncsu.edu.
authors.forename: Flora
authors.initials: F
authors.lastname: Meilleur
mesh_terms: D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009492:Neurospora crassa / Q000201:enzymology*; D010084:Oxidation-Reduction; D053838:Scattering, Small Angle
chemical_list: D006899:Mixed Function Oxygenases; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase
keywords: Cellobiose dehydrogenases; Neutron diffraction; Neutron scattering; Polysaccharide monooxygenases; X-ray induced photoreduction