Molecular-Level Origins of Biomass Recalcitrance: Decrystallization Free Energies for Four Common Cellulose Polymorphs

Abstract

Cellulose is a crystalline polymer of β1,4-D-glucose that is difficult to deconstruct to sugars by enzymes. The recalcitrance of cellulose microfibrils is a function of both the shape of cellulose microfibrils and the intrinsic work required to decrystallize individual chains, the latter of which is calculated here from the surfaces of four crystalline cellulose polymorphs: cellulose Iβ, cellulose Iα, cellulose II, and cellulose III(I). For edge chains, the order of decrystallization work is as follows (from highest to lowest): Iβ, Iα, ΙΙΙ(Ι), and II. For cellulose Iβ, we compare chains from three different locations on the surface and find that an increasing number of intralayer hydrogen bonds (from 0 to 2) increases the intrinsic decrystallization work. From these results, we propose a microkinetic model for the deconstruction of cellulose (and chitin) by processive enzymes, which when taken with a previous study [Horn et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18089] identifies the thermodynamic and kinetic attributes of enzyme and substrate engineering for enhanced cellulose (or chitin) conversion. Overall, this study provides new insights into the molecular interactions that form the structural basis of cellulose, which is the primary building block of plant cell walls, and highlights the need for experimentally determining microfibril shape at the nanometer length scale when comparing conversion rates of cellulose polymorphs by enzymes.

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

“…Subsequent O-linked glycans were bonded as α-1,2 linkages (47). The cellulose Iβ microfibril was modeled based on an equilibrated slab from a previous study (48), which was initially constructed from the cellulose Iβ crystal structure (49). The cellulose microfibril is 28 glucose units long with four layers of cellulose chains.…”

“…The top layer of cellulose comprises three chains. The TrCel7A CD was built with an edge chain in the CD tunnel, because edge chains are easier to decrystallize than middle chains from the hydrophobic surface (48). The CBM and CD were placed on the hydrophobic face of cellulose, because this is the preferred binding face for the CBM (12).…”

“…The linker was constructed with two O-linked mannose residues per glycosylation site, which is an assumed glycosylation pattern based on the known TrCel7A linker glycosylation (26). Because the TrCel6A linker is longer than the TrCel7A linker by 15 residues, the cellulose Iβ microfibril required a larger binding surface (48), which was constructed to be 36 glucose units long with four layers of cellulose chains. As with TrCel7A, the top layer of cellulose comprised three chains.…”

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

“…The enzyme was placed on the hydrophobic surface at the nonreducing end. The cellodextrin chain from the crystal was bonded to the ligand at the ϩ4 position manually, and the threaded chain was an "edge" chain, which are easier to decrystallize relative to "middle" chains (13). The positions of the cellobiose and the cellotetraose bound at the tunnel were the same as in the TfuCel6B-cellodextrin system.…”

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

“…Adsorption of the enzymes on the surface of the substrate is the initial step, and then the polysaccharide chain is loaded into the active site tunnel or cleft of the catalytic module, the glycosidic bond is cleaved by hydrolysis, the reaction product is released and the catalytic module slides along the polymer substrate to the next cleavage site. The reactions are heterogeneous, and the enzymes acting at a solid-liquid interface need to overcome the decrystallization energy during the process [4][5][6] . Although vast numbers of glycanases are predicted to work processively, it has been difficult to evaluate processivity by means of biochemical experiments, and therefore the structure-function relationship of processive glycanases is poorly understood.…”