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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Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.

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