Computational analysis of cellobiohydrolase Cel7b from Melanocarpus albomyces

Abstract

Cellulases are a broad class of enzymes responsible for decomposing lignocellulosic biomass. These enzymes have several industrial applications, most notably in the production of 2nd generation biofuels from cellulosic feedstock. However, current degradation technologies involving cellulase cocktails are not sufficiently optimized for economical biofuel production on an industrial scale. Thus, there is significant incentive to further understand and improve the enzymatic degradation of cellulose by cellulase enzymes. In this thesis, constant pH molecular dynamics simulations (CpHMD), classical molecular dynamics (MD), docking calculations and kinetic modeling were utilized to evaluate the fundamental interactions impacting cellulose degradation by the cellobiohydrolase Cel7B from Melanocarpus albomyces (Ma). Presented in this thesis is an extensive evaluation of the ionizable residues in [alpha]-Conotoxin by both CpHMD and [superscript 1]H NMR, which serves as a validation of the computational pKa prediction procedures used in the subsequent thesis chapters. The procedures established in the study of [alpha]-Conotoxin were then utilized in the simulations of active site residues in Ma Cel7B. The pKa values of active site residues predicted by the simulations support the role of Glu217 as the catalytic acid-base and Glu212 as the catalytic nucleophile. In addition to predicting pKa values, the simulations identified significant charge correlations and hydrogen bonding networks that are critical to hydrolysis of the glycosidic bond. The results from the CpHMD simulations were then incorporated into a kinetic model, which further supports the hypothesis that hydrogen bonding and charge coupling are needed to achieve an optimal activity near the experimental active pH of Ma Cel7B. Beyond residue pKa values and their influence on the observed enzymatic rate, standard MD and CpHMD simulations were used to evaluation protein dynamics and loop flexibility. Investigation of peripheral loops enclosing the active site revealed structural fluctuations that are likely crucial to the binding and threading of the cellulose polymer substrate, as well as contributing to the pH and temperature tolerance of Ma Cel7B. It was found that the protonation of several residues on adjacent peripheral loops are responsible for the observed loop fluctuations and overall conformation in the free enzyme. Simulations with substrate bound in the active site reveal significant changes in the conformation and fluctuation patterns of several peripheral loop regions. The substrate induced response of the loop regions secures the cellulose polymer in the catalytic tunnel, creating an environment that is conducive for hydrolysis of the glycosidic bond. Similar loop fluctuations and dynamics are also observed when a free enzyme resides on a cellulose microfibril, indicating the role of the peripheral loops in guiding substrate into the catalytic tunnel. To further probe enzyme-substrate interactions on the hydrolysis of cellulose, the confirmation of the sugar ring at the catalytic site was investigated under different residue protonation environments. In general, the results indicate the highly charge coupled active site effectively modulates the formation of the catalytically active skewed-boat confirmation, and clearly identifies the protonation states of active site residues as the major contributing factor to the formation of the skewed boat configuration. The results presented in this thesis provide insights into molecular-level interactions that lead to the observed enzyme characteristics of Ma Cel7B, and indicate computational methods can be used to gain valuable insights into the protonation environment and specific residue pKa values that are crucial to the hydrolysis reaction performed by family 7 cellulase enzymes.