Cellobiose Dehydrogenase and a Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation by Neurospora crassa

Abstract

The high cost of enzymes for saccharification of lignocellulosic biomass is a major barrier to the production of second generation biofuels. Using a combination of genetic and biochemical techniques, we report that filamentous fungi use oxidative enzymes to cleave glycosidic bonds in cellulose. Deletion of cdh-1, the gene encoding the major cellobiose dehydrogenase of Neurospora crassa, reduced cellulase activity substantially, and addition of purified cellobiose dehydrogenases from M. thermophila to the Δcdh-1 strain resulted in a 1.6- to 2.0-fold stimulation in cellulase activity. Addition of cellobiose dehydrogenase to a mixture of purified cellulases showed no stimulatory effect. We show that cellobiose dehydrogenase enhances cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of copper-dependent polysaccharide monooxygenases (PMOs) that catalyze the insertion of oxygen into C-H bonds adjacent to the glycosidic linkage. Three of these PMOs were characterized and shown to have different regiospecifities resulting in oxidized products modified at either the reducing or nonreducing end of a glucan chain. In contrast to previous models where oxidative enzymes were thought to produce reactive oxygen species that randomly attacked the substrate, the data here support a direct, enzyme-catalyzed oxidation of cellulose. Cellobiose dehydrogenases and proteins related to the polysaccharide monooxygenases described here are found throughout both ascomycete and basidiomycete fungi, suggesting that this model for oxidative cellulose degradation may be widespread throughout the fungal kingdom. When added to mixtures of cellulases, these proteins enhance cellulose saccharification, suggesting that they could be used to reduce the cost of biofuel production.

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

“…Although CBM33-type and GH61-type LPMOs seem to catalyze essentially the same reaction (4,5,12,13,23) and have similar active sites ( Fig. 1 D and E), their metal binding sites do show some notable differences.…”

“…1). The identity of the metal was not disclosed in early work (5,10), but functional studies on related enzymes, a CBM33 from Enterococcus faecalis (11) and three GH61s (4,12,13), have later indicated that the LPMOs are copper-dependent enzymes. However, because of several complications, such as the indiscriminate nature of the metal binding site (see below) and the fact that the CBM33 crystal structures obtained so far mostly were metal-free, many aspects of metal binding remain unresolved.…”

“…Ref. 4 discusses additional suggestions concerning the mechanism. Note that copper binding involves three nitrogen atoms, as indicated, namely the N-terminal amino group and an imidazole nitrogen in each of the two conserved histidines.…”

Spectroscopic and computational insight into the activation of O 2 by the mononuclear Cu center in polysaccharide monooxygenases

“…It is interesting to note that in a recently published study by Isaksen et al (38), hydrogen peroxide formation, by a Cu-AA9 enzyme with different reductants and O 2 , was detected only in the absence of substrate or with substrates that were not subject to AA9 activity. If the superoxide is indeed stabilized by the substrate, it may be able to directly attack the polysaccharide, or it may be further reduced to a more reactive species by either small molecules or cellobiose dehydrogenase (a known AA9 reducing cofactor) (7,13,39). This awaits further experimental investigation.…”

“…Recently, a class of oxygen activating enzymes with a single copper center has been identified, the polysaccharride monooxygenases [PMOs; often termed lytic polysaccharide monooxygenases (LPMOs), reflecting their ability to break polysaccharides chains and loosen crystalline structure] (6-8), or AA9 to 11 enzymes (AA = auxiliary activity) in the Carbohydrate-Active enZYmes (CAZy) database (9). AA9 enzymes [formerly glycoside hydrolases family 61 (GH61s)] are fungal enzymes that can enhance major cellulases' enzymatic degradation of cellulose (hence "auxiliary activity"), whereas AA10 enzymes [formerly carbohydrate binding module family 33 (CBM33)] are predominantly bacterial enzymes that can enhance major chitinases' degradation of chitin (6,7,(10)(11)(12)(13). Enzymes in the latest discovered subclass, AA11, are fungal enzymes, where the currently lone characterized example uses chitin as a substrate (8).…”