Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

Cost-effective enzymatic breakdown of recalcitrant polysaccharides such as cellulose and chitin to constituent sugars has tremendous potential for industrial applications for the production of fuels and high-value chemicals. Research towards that goal has been aided recently by the discovery and classification of polysaccharide mono-oxygenase enzymes (GH61s), which conduct oxidation of glycosidic linkages and thus represent a new enzymatic paradigm in carbohydrate deconstruction from the standard glycoside hydrolases. Recent experimental studies suggest that GH61s cleave chains oxidatively in crystalline regions of cellulose and chitin, exposing further ends for processive degradation, which leaves an oxidized end exposed to solution. Using molecular dynamics and free energy calculations, we explore what impact potential oxidations have on the overall structure of the cellulose crystal, including their impact on the free energy of fibril decrystallization. Particularly noteworthy differences appear in the solvent-accessible surface area and decrystallization free energy changes of particular oxidation states, suggesting that these states are more accessible to processive cellulose-degradation enzymes. In addition, the soluble deconstruction products of oxidation will potentially bind to the active sites of cellulases. To examine this effect, we have used thermodynamic cycles to compute ΔΔG between the hydrolyzed and oxidized products in the active site of the Family 7 and Family 6 glycoside hydrolases from Trichoderma reesei, which are key industrial enzymes and commonly used model systems for fungal cellulases. Our analysis shows that oxidation brings about changes in the hydrogen bonding pattern within the cellulases, and thus changing the binding affinity and product inhibition depending on the oxidation state.