Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

In
nature, polysaccharide glycosidic bonds are cleaved by hydrolytic
enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO).
These oxidative enzymes are synergistic with cocktails of hydrolytic
enzymes and are thought to act primarily on crystalline regions, in
turn providing new sites of productive attachment and detachment for
processive hydrolytic enzymes. In the case of cellulose, the homopolymer
of β-1,4-d-glucose, enzymatic oxidation occurs at either
the reducing end or the nonreducing end of glucose, depending on enzymatic
specificity, and results in the generation of oxidized chemical substituents
at polymer chain ends. LPMO oxidation of cellulose is thought to produce
either a lactone at the reducing end of glucose that can spontaneously
or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing
end that may further oxidize to a geminal diol. Here, we use molecular
simulation to examine the effect of oxidation on the structure of
crystalline cellulose. The simulations highlight variations in behaviors
depending on the chemical identity of the oxidized species and its
location within the cellulose fibril, as different oxidized species
introduce steric effects that disrupt local crystallinity and in some
cases reduce the work needed for polymer decrystallization. Reducing-end
oxidations are easiest to decrystallize when located at the end of
the fibril, whereas nonreducing end oxidations readily decrystallize
from internal cleavage sites despite their lower solvent accessibility.
The differential in decrystallization free energy suggests a molecular
mechanism consistent with experimentally observed LPMO/cellobiohydrolase
synergy. Additionally, the soluble oxidized cellobiose products released
by hydrolytic cellulases may bind to the active sites of cellulases
with different affinities relative to cellobiose itself, which potentially
affects hydrolytic turnover through product inhibition. To examine
the effect of oxidation on cello–oligomer binding, we use thermodynamic
integration to compute the relative change in binding free energy
between the hydrolyzed and oxidized products in the active site of
Family 7 and Family 6 processive glycoside hydrolases, Trichoderma
reesei Cel7A and Cel6A, which are key industrial cellulases
and commonly used model systems for fungal cellulases. Our results
suggest that the equilibrium between the two reducing end oxidized
products, favoring the linear aldonic acid, may increase product inhibition,
which would in turn reduce processive substrate turnover. In the case of LMPO action at the nonreducing end, oxidation appears to lower affinity
with the nonreducing end specific cellulase, reducing product inhibition
and potentially promoting processive cellulose turnover. Overall,
this suggests that oxidation of recalcitrant polysaccharides by LPMOs
accelerates degradation not only by increasing the concentration of
chain termini but also by reducing decrystallization work, and that
product inhibition may be somewhat reduced as a result.