Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.

Significance: Plant cell walls contain significant amounts of the polysaccharides cellulose and hemicellulose, which can be depolymerized by enzymes to sugars and upgraded to renewable fuels and chemicals. Traditionally, enzymes for biomass depolymerization were based on naturally occurring hydrolytic enzymes, until the recent discovery of another natural enzymatic paradigm for carbohydrate deconstruction. Namely, lytic polysaccharide monooxygenases (LPMOs), long thought to be hydrolases or carbohydrate-binding modules, were revealed to be oxidative, copper-containing enzymes. These enzymes are receiving significant attention as they could revolutionize biomass deconstruction to upgradeable intermediates for renewable energy applications. Here, we apply quantum mechanical calculations to elucidate the oxidative reaction mechanism to offer predictions into how LPMOs function.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major im-portance for efficient processing of biomass. NcLP-MO9C from Neurospora crassa acts both on cellu-lose and on non-cellulose β-glucans, including cello-dextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extend-ed highly polar substrate-binding surface well-suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interac-tions. Electron spin resonance (EPR) studies demon-strated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry (ITC) studies revealed binding affinities in the low micromolar range for polymeric substrates that are in part due to the presence of a carbohydrate-binding module (a CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4 or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1 oxidizing LPMO9s, access to the sol-vent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4 oxidizing LPMO9s. LPMO9s known to produce a mixture of C-1 and C4-oxidized products show an intermediate situation.