The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of “cellulase-enhancing factors,” such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes.

The emerging bioeconomy depends on improved methods for processing of lignocellulosic biomass to fuels and chemicals. Saccharification of lignocellulose to fermentable sugars is a key step in this regard where enzymatic catalysis plays an important role and is a major cost driver. Traditionally, enzyme cocktails for the conversion of cellulose to fermentable sugars mainly consisted of hydrolytic cellulases. However, the recent discovery of lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose using molecular oxygen and an electron donor, has provided new tools for biomass saccharification. Current commercial enzyme cocktails contain LPMOs, which, considering the unique properties of these enzymes, may change optimal processing conditions. Here, we show that such modern cellulase cocktails release up to 60 % more glucose from a pretreated lignocellulosic substrate under aerobic conditions compared to anaerobic conditions. This higher yield correlates with the accumulation of oxidized products, which is a signature of LPMO activity. Spiking traditional cellulase cocktails with LPMOs led to increased saccharification yields, but only under aerobic conditions. LPMO activity on pure cellulose depended on the addition of an external electron donor, whereas this was not required for LPMO activity on lignocellulose. In this study, we demonstrate a direct correlation between saccharification yield and LPMO activity of commercial enzyme cocktails. Importantly, we show that the LPMO contribution to overall efficiency may be large if process conditions are adapted to the key determinants of LPMO activity, namely the presence of electron donors and molecular oxygen. Thus, the advent of LPMOs has a great potential, but requires rethinking of industrial bioprocessing procedures.

Significance: Activation of the O-O bond in dioxygen is difficult but fundamental in biology. Nature has evolved several strategies to achieve this, often including copper as an enzyme cofactor. Copper-dependent enzymes usually use more than one metal to activate O ₂ by multielectron reduction, but recently it was discovered that cellulose and chitin degrading polysaccharide monooxygenase enzymes use only a single Cu center for catalysis, in a reaction that is of great interest to the biofuel industries. To understand this reactivity, we have determined the solution structures of both the reduced and oxidized Cu site, and determined experimentally and computationally how this site is capable of facile O ₂ activation by a thermodynamically difficult one-electron reduction, via an inner-sphere Cu-superoxide intermediate.

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.