Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. biofuels | cellulase | post-translational modification | carbohydrate recognition

No abstract

The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions. As shown by ESI-MS and PAG-IEF analyses of both intact protein and the isolated catalytic core module, the microheterogeneity originates mainly from the variable ratio of single N-acetylglucosamine over high-mannose structures on the three N-glycosylation sites and from the presence or absence of phosphate residues. Fully N- and O-glycosylated Cel7A can only be isolated from minimal medium and probably reflects the initial complexity of the protein on leaving the glycosynthetic pathway. Extracellular activities are responsible for postsecretorial modifications in other cultivation conditions: alpha-(1-->2)-mannosidase, alpha-(1-->3)-glucosidase and an Endo H type activity participate in N-deglycosylation (core), whereas a phosphatase and a mannosidase are probably responsible for hydrolysis of O-glycans (linker). The effects are most prominent in corn steep liquor-enriched media, where the pH is closer to the pH optimum (5-6) of these extracellular hydrolases. In minimal medium, the low pH and the presence of proteases could explain for the absence of such activities. On the other hand, phosphodiester linkages in the catalytic module are only observed under specific conditions. The extracellular trigger is still unknown, but mannophosphorylation may be regulated intracellularly by alpha-(1-->2)-mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.

We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-bridge” hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating.