Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper–oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

“…A detailed account of the experimental procedure, data collection and refinement strategy has been described. 80 Provided here are the experimental details in brief as well as descriptions of the computational methods employed.…”

Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography

“…The exact mechanism by which LPMOs achieve catalysis remains to be elucidated and the poorly soluble carbohydrate substrate presents challenges in crystallographic structural analysis, although a limited number of structures have been determined (Frandsen et al, 2016;Simmons et al, 2017;Tandrup et al, 2020). Future spectroscopic, computational and structural studies, including neutron protein diffraction studies investigating the NcLPMO9D structure under functionally relevant acidic conditions and bound to a cryo-trapped intermediate (Schrö der et al, 2021), may help to shed light on LPMO catalysis.…”

Toward elucidating the mechanism of lytic polysaccharide monooxygenases: chemical insights from X-ray and neutron crystallography

“…Neutron cryo-crystallography permits trapping of catalytic intermediates, thereby providing insight into protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To this end, we collected a cryo-neutron diffraction dataset on an ascorbate-reduced LPMO9D crystal to characterize the reaction mechanism intermediates (Schröder et al 2021). A second neutron diffraction dataset was collected at room temperature on a LPMO9D crystal exposed to low pH conditions to probe protonation states under acidic conditions.…”