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...

Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization.

Abstract:The economic utilization of abundant lignocellulosic biomass as a feedstock for the production of fuel and chemicals would represent a profound shift in industrial carbon utilization, allowing sustainable resources to substitute for, and compete with, petroleum based products. In order to exploit biomass as a source material for production of renewable compounds, it must first be broken down into constituent compounds, such as sugars, that can be more easily converted in chemical and biological processes. Lignocellulose is, unfortunately, a heterogeneous and recalcitrant material which is highly resistant to depolymerization. Many microorganisms have evolved repertoires of enzyme activities which act in tandem to decompose the various components of lignocellulosic biomass. In this review, we discuss recent advances in the understanding of these enzymes, with particular regard to those activities deemed likely to be applicable in commercialized biomass utilization processes.

The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s ؊1 at 30°C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

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