Structural Investigations of the Fungal Lytic Polysaccharide Monooxygenase Reaction Mechanism using X-ray and Neutron Protein Crystallography

Industrial production of biofuels and other value-added products derived from cellulosic feedstocks relies on enzymatic hydrolysis of cellulose to convert biomass to soluble sugars. The overall conversion efficiency and the cost of final products are influenced greatly by the efficiency of hydrolases which is diminished by cellulose recalcitrance arising from high degrees of polymerization, crystallinity and intra-/intermolecular hydrogen bonding. Efforts over the last 20 years to discover more efficient hydrolases have instead identified oxidative enzymes that boost the efficiency of cellulose conversion. These lytic polysaccharide monooxygenases (LPMOs) break cellulose chains by oxidizing carbon atoms involved in glycosidic bonds. LPMOs do not directly release soluble sugars but instead function as an important adjuvant to hydrolase function that increases industrial conversion efficiency by one or more orders of magnitude.;LPMOs are mononuclear copper metalloenzymes dependent upon an input of reducing equivalents that activate molecular O2 for the net insertion of an oxygen atom into a carbon-- hydrogen bond. Herein are described important new structural characterizations of this reactivity derived from crystallographic studies of the enzyme Neurospora crassa LPMO9D in resting and O2-activated states. High resolution X-ray cryocrystallography has revealed that fungal LPMOs activate O2 as an equatorial ligand of the active site Cu(I/II) ion. Molecular O 2 activation likely follows an initial "pre-binding" step in which molecular O2 occupies a polar binding site adjacent to the active site ion. Analysis of the first reported LPMO structure determined by neutron protein crystallography provided new details of protonation states and hydrogen bonding around the active site and indicated a role of a conserved histidine in stabilizing "pre-bound" O2. This role has been confirmed with quantum calculations. The impacts of this research for current mechanistic understanding and future experiments are also discussed.