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.ellulose is Earth's most abundant biopolymer. Its exploitation as an energy source plays a critical role in the global ecology and carbon cycle. Industrial production of fuels and chemicals from this plentiful and renewable resource holds the potential to displace petroleum-based sources, thus reducing the associated economic and environmental costs of oil and gas production (1, 2) and promoting energy security as part of a balanced energy portfolio. However, despite the burgeoning potential of cellulose as a biofuel source, its remarkable recalcitrance to depolymerization has so far hindered the economical use of any form of lignocellulosic biomass as a feedstock for biofuel production (3, 4).In addressing the issue of cellulose recalcitrance, much effort has been directed toward harnessing the known cellulosedegrading enzymatic pathways found in fungi. The consensus model of enzymatic degradation involves the concerted action of a consortium of different endoglucanases and "exo"-acting cellobiohydrolases (collectively termed "cellulases"); both enzyme classes perform classical glycoside hydrolysis through attack of water at the anomeric center of oligo/polysaccharide substrates (5-9). Necessarily as part of the overall enzymatic degradation of cellulose, the initial enzymatic step must overcome cellulose's inertness by disrupting the cellulosic structure, thus allowing attack by traditional cellulases. Originally, Reese et al. (10) suggested that undefined enzymes could play a major role in this step. This notion remained a hypothesis until very recently when, in a key paper, Harris et al. (11) demonstrated that inclusion of a novel enzyme class, currently termed GH61 glycoside hydrolases in the CAZy database of carbohydrate-active enzymes (12), greatly increases the performance of Hypocrea jecorina (Trichoderma reesei) cellulases in lignocellulose hydrolysis. From this work, it was suggested that GH61s act directly on cellulose rendering it more accessible to traditional cellulase action (11). Moreover, recent genomi...

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Strategies for O 2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O 2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O 2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O 2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O 2 binding with very little reorganization energy.X-ray absorption spectroscopy | DFT | dioxygen activation | biofuels

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.