Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: A docking study
ABSTRACT Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.
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ABSTRACT: AutoDock is a small-molecule docking program that uses an energy function to score docked ligands. Here AutoDock's grid-based method for energy evaluation was exploited to evaluate the force exerted by Fusarium oxysporum Cel7B on the atoms of docked cellooligosaccharides and a thiooligosaccharide substrate analog. Coupled with the interaction energies evaluated for each docked ligand, these forces give insight into the dynamics of the ligand in the active site, and help to elucidate the relative importance of specific enzyme-substrate interactions in stabilizing the substrate transition-state conformation. The processive force on the docked substrate in the F. oxysporum Cel7B active site is less than half of that on the docked substrate in the Hypocrea jecorina Cel7A active site. Hydrogen bonding interactions of the enzyme with the C2 hydroxyl group of the glucosyl residue in subsite -2 and with the C3 hydroxyl group of the glucosyl residue in subsite +1 are the most significant in stabilizing the distorted14B transition-state conformation of the glucosyl residue in subsite -1. The force calculations also help to elucidate the mechanism that prevents the active site from fouling.Proteins Structure Function and Bioinformatics 11/2005; 61(3):590-6. DOI:10.1002/prot.20632 · 2.92 Impact Factor
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ABSTRACT: Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.Biotechnology Journal 05/2007; 2(5):546-58. DOI:10.1002/biot.200600221 · 3.71 Impact Factor