Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Abstract

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

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

“…The affinity values achieved for the nonglycosylated CBM 1 are in agreement with other studies (21,45,51). We report affinity enhancements of similar magnitudes as the enhancements reported by Takashima et al (50) and Linder et al (12), with affinity of CBMs 13 and 17 approaching or within range of K ads values obtained for both the whole TrCel7A or the multiglycosylated TrCel7A linker CBM domain (21,(52)(53)(54)(55), suggesting that glycoengineering of Family 1 CBMs is a viable strategy for activity enhancement of cellulases.…”

“…The binding affinity of the TrCel7A CBM glycoforms to crystalline cellulose was also measured using a similar method to our previous studies (21). The binding affinity of each CBM glycoform for bacterial microcrystalline cellulose (BMCC) was fitted to a Langmuir isotherm and is reported as K ads , which correlates with the strength of CBM adsorption to BMCC.…”

“…Glycosylation can modulate both the physical and biological properties of proteins (39,40) and aid in protein folding and secretion (41). Indeed, O-linked glycans on cellulase linkers confer proteolytic resistance (20,42) and have been shown to impart affinity to crystalline cellulose (21). Furthermore, it has been shown that small O-linked glycans exist on the TrCel7A CBM near the binding face ( Fig.…”

“…To date, few studies have been conducted to examine glycosylation in secreted fungal enzymes to determine the extent and factors that control it, including growth conditions and extracellular glycan-trimming enzymes. Catalytic domains can exhibit both N-and O-linked glycans (18,19), whereas the linkers connecting enzymatic domains to CBMs are decorated with O-linked glycosylation, which has long been attributed to protease protection (20) and more recently implicated in substrate binding (21). For TrCel7A, Harrison et al (22) published the original characterization of the glycosylation pattern on the TrCel7A linker.…”

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

“…System minimization was conducted in two steps: 1000 steps of steepest descent, followed by 1000 steps of conjugate gradient (Payne et al, 2013). Minimization was followed by short simulation for 10 ps with positional restraint on protein molecules.…”