Molecular dynamics (MD) simulations will be performed to gain fundamental understanding of the function of two important biomass-degrading enzymes, β-glucosidases (βgls) and lytic polysaccharide monooxygenases (LPMOs). The calculations will complement structural and biochemical studies conducted by the group.
βgls relieve endoglucanase and cellobiohydrolase product inhibition through hydrolysis of cellobiose to glucose. However, their catalytic activity is significantly reduced at high cellobiose or glucose concentration owing to their susceptibility to transglycosylation, a competing pathway wherein a sugar, instead of water, attacks the glycosyl-enzyme intermediate to form a di- or trisaccharide. It is hypothesized that one of the important governing factors in the balance between the two pathways is the effect of the active environment on the relative stabilities of their corresponding transition states (TSs). Thus, hydrolysis and transglycosylation (as well as the preceding glycosylation step) will be modeled using quantum mechanics/molecular mechanics umbrella sampling (QM/MM-US). Glycoside Hydrolase Family 1 (GH1) and Family 3 (GH3) βgls will both be examined because they possess distinct active site environments. The acid/base residue in GH3 βgl is flanked by two arginine residues, while that in GH1 βgl is surrounded by hydrophobic residues. Moreover, a tyrosine residue near the nucleophile is believed to affect the character of the hydrolysis TS in GH1 βgl through hydrogen bonding. This interaction is unlikely in GH3 βgl because of the different position of the corresponding tyrosine. The data will provide information on the subtle differences in the character of the hydrolysis and transglycosylation TSs and the active site residues critical to the stability of each TS.
LPMOs are copper-containing oxygenases that have been recently attracting much attention for their capability of acting on recalcitrant substrates such as cellulose and chitin. Polysaccharide chains at the surface of the crystalline polymer are cleaved upon oxidation by LPMOs, facilitating further degradation of the substrate by other enzymes. The mechanism of action of LPMOs remains largely unknown. This includes the binding mode of the substrate, which would have an impact on substrate specificity (cellulose, hemicellulose, or chitin) and regiospecificity of oxidative attack (C1- or C4-position). Knowledge of the mechanism is limited owing to the challenges involved in performing experiments with insoluble substrates. To date, the only available crystal structures of LPMO with bound substrate is that of the Auxiliary Activity Family 9 (AA9) LPMO from Lentinulis similis with cellotriose and cellohexaose. It is hypothesized that the length and residue composition of the loop regions near the active site, which are not highly conserved, is a major determinant of substrate binding. This will be investigated through an extensive molecular dynamics (MD) study of AA9, AA Family 10 (AA10), AA Family 11 (AA11) LPMOs bound to various model oligosaccharides representing cellulose, hemicellulose, or chitin. The data will provide insight on structural changes in the copper active site upon substrate binding and how differences in loop composition and dynamics lead to the variation in substrate specificity and regiospecificity exhibited by LPMOs.