Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering. (7th August 2017) - Bodenheimer, Annette M.
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 ( Nc LPMO9D) and CDHIIA ( Nc CDHIIA), 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 Nc LPMO9D 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 Nc CDHIIA 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. Graphical abstract: Highlights: Neutrons allow structural studies of cellulose degradation oxidative enzymes without X-ray induced photoreduction. Neutron crystallography reveals the protonation states and hydrogen bond interactions at the active site of Nc LPMO9D. Small-angle neutron scattering confirms that Nc CDHIIA is extended in the oxidized state.