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...

BLUF-domain-comprising photoreceptors sense blue light by utilizing FAD as a chromophore. The ycgF gene product of Escherichia coli is composed of a N-terminal BLUF domain and a C-terminal EAL domain, with the latter postulated to catalyze c-di-GMP hydrolysis. The linkage between these two domains involves a predominantly helical segment. Its role on the function of the YcgF photoreceptor domain was examined by characterizing BLUF domains with and without this segment and reconstituting them with either FAD, FMN or riboflavin. The stability of the light-adapted state of the YcgF BLUF domain depends on the presence of this joining, helical segment and the adenosine diphosphate moiety of FAD. In contrast to other BLUF domains, two-dimensional (1)H,(15)N and one-dimensional (1)H NMR spectra of isotope-labeled YcgF-(1-137) revealed large conformational changes during reversion from the light- to the dark-adapted state. Based on these results the function of the joining helix in YcgF during signal transfer and the role of the BLUF domain in regulating c-di-GMP levels is discussed.

SUMMARYTo explain the decline of Hippopha~ scrub in the vegetation succession in the dunes of The Netherlands, the growth and nodulation of Hippopha~ plants grown in pots, using soil from an early stage (site AH) and a post-optimum stage (site HP), were investigated. In HP-soil nodulation, yield, and the nitrogen and phosphorus content of test plants were always lower and the number of necrotic nodules and the dry matter content were always higher than in AH-soil, even after inoculation with crushed nodules and the addition of a nutrient solution. Plants in HP-soil also had darker roots, less root hairs, a higher number of short lateral roots and a higher percentage of dead roots than those in AH-soil. These characteristics of adverse growth conditions disappeared upon ignition or gammairradiation of HP-soil.Possible explanations of these results are discussed. The degeneration of Hippophai5 scrub cannot be ascribed to the age of the plants, the absence of sutticient infective endophyte particles or to abiotic factors such as unfavourable physical (particle size) or chemical soil conditions but is caused by biotic factors. No indications were obtained that plant-pathogenic fungi and bacteria are involved. HP-soil in contrast to AH-soil, however, contained large numbers of the nematode Longidorus sp., a species known to cause root deformations. The conclusion was that this nematode is one of the biotic factors involved in the degeneration of the Hippophai~ scrub. This degeneration is due to a restriction of the root system resulting in a low phosphate uptake, a low nodulation capacity and, as a consequence, a low nitrogen content. The results demonstrate that biotic soil factors are important in influencing succession in higher plant communities.

Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F 1 -ATPase data set and a 4.5 Å resolution SecYEG-SecA complex data set. All of the models were automatically built by the method to R free values of between 28.9 and 39.9% and were free from the initial model bias.

Juvenile plants of Plantago lanceolata and P. major ssp. major were grown on nutrient solution at optimal availability of nitrate as well as at various degrees of suboptimal nitrate availability. In the optimal treatment the nutrient solution contained 7.5 mmol NO per 1. In the various suboptimal treatments nitrate of the basic solution was replaced by sulphate, and nitrate was continually added during the experimental period. The rate of addition was exponential, according to the formula: [Formula: see text] (N=initial content of nitrogen in the seedling, RAR=relative addition rate, t=time in days). There were four suboptimal treatments corresponding to RAR's of 0.25, 0.20, 0.15 and 0.10 per day. In all cases plants were in steady state. The plant parameters as varied in the experimental treatments were related to the N concentrations (% fresh weight). Most relationships were linear. P. major attained a higher RGR than P. lanceolata at equal N concentrations. The root weight ratio was inversely proportional to the N concentration, and varied between 0.55 and 0.25. The N productivity (mg·(mg N)·d) with respect to shoot biomass was proportional to the N concentration, and higher in P. major. This difference is related to the lower N concentration of P. major. The N productivity with respect to root biomass was highest when the N concentration was reduced by about 50 per cent. The length of the root axis was inversely proportional to the N concentration, and greater in P. lanceolata. The ecological implications of the experimental results were discussed.