Molecular Persistent Cellulase Product Release Process Dynamics Simulation

Understanding the enzymatic mechanism that cellulasesemploy to degrade cellulose is critical to efforts to efficientlyutilize plant biomass as a sustainable energy resource. As arepresentativefor investigating the enzymatic mechanism of cellulases, Cellobiohydrolase I of Trichoderma reesei(Hypocrea jecorina) has the ability to hydrolysecrystalline cellulose in a processive manner. The processive cycle of CBHI includes3steps, namely hydrolysis, product release and cellulose threading, while product release is the key step of the cycle to connect hydrolysis and cellulose threading, because product inhibition could result in a break of the processive cycle and asharp reduction of enzyme activity. In order to explore CBHI product release process at atomic level, firstly we constructed CBHI product release model where the product cellobiose bindsto the product sites of catalysis tunnel, and then replica exchange molecular dynamics (REMD) simulations wereconducted for128temperature replicas with250ns simulation for each replica atNational Supercomputer Center in Jinan, and then data analysis was done at Shandong High Performance Computing Center.Analysis of the REMD trajectories suggests that there are3popular release pathways(W376<-(?)H228, Q175(?)H228and R251(?)H228) through which product move out of the catalytic tunnel fromproduct sites.Analysis of CBHI tunnel loops dynamics shows that loopⅢ and loopⅣ can open during product release process, which implies that loop motions are important to product release.Predicted residues175GLN,228HIS,251ARG,259ASP and376TRPmay play key roles in cellobiose release. The binding free energy of cellobiose binds to CBHI active sites is-62.134kJ/mol at300K by MM/GBSA method, which means that product inhibition isthermodynamicallyfavorable.Based on cellobiose binding free energy and Arrhenius equation,the calculated product release rate16s-1is in good agreement with AFM observation, which implies that product release maybe the rate-limiting step of CBHI processive motion.Overall, this study reveals new structural and dynamical details of CBHI product release processand predicts key regions and residues for product release, which provides potential applications in engineering the enzyme to accelerate the product release process and improve the efficiency of biomass conversion.