Katja Salomon Johansen

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components

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 coppers coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

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.

Characterization and Three-Dimensional Structures of Two Distinct Bacterial Xyloglucanases from Families Gh5 and Gh12.

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed “hemicellulose.” One such hemicellulose is xyloglucan, which displays a β-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (β/α)8 and β-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the β-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (β/α)8 GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases

Significance Activation of the O-O bond in dioxygen is difficult but fundamental in biology. Nature has evolved several strategies to achieve this, often including copper as an enzyme cofactor. Copper-dependent enzymes usually use more than one metal to activate O2 by multielectron reduction, but recently it was discovered that cellulose and chitin degrading polysaccharide monooxygenase enzymes use only a single Cu center for catalysis, in a reaction that is of great interest to the biofuel industries. To understand this reactivity, we have determined the solution structures of both the reduced and oxidized Cu site, and determined experimentally and computationally how this site is capable of facile O2 activation by a thermodynamically difficult one-electron reduction, via an inner-sphere Cu-superoxide intermediate. Strategies for O2 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 O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 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 O2 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 O2 binding with very little reorganization energy.

Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme.

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

Low temperature lignocellulose pretreatment: effects and interactions of pretreatment pH are critical for maximizing enzymatic monosaccharide yields from wheat straw

BackgroundThe recent development of improved enzymes and pentose-using yeast for cellulosic ethanol processes calls for new attention to the lignocellulose pretreatment step. This study assessed the influence of pretreatment pH, temperature, and time, and their interactions on the enzymatic glucose and xylose yields from mildly pretreated wheat straw in multivariate experimental designs of acid and alkaline pretreatments.ResultsThe pretreatment pH was the most significant factor affecting both the enzymatic glucose and xylose yields after mild thermal pretreatments at maximum 140°C for 10 min. The maximal enzymatic glucose and xylose yields from the solid, pretreated wheat straw fraction were obtained after pretreatments at the most extreme pH values (pH 1 or pH 13) at the maximum pretreatment temperature of 140°C. Surface response models revealed significantly correlating interactions of the pretreatment pH and temperature on the enzymatic liberation of both glucose and xylose from pretreated, solid wheat straw. The influence of temperature was most pronounced with the acidic pretreatments, but the highest enzymatic monosaccharide yields were obtained after alkaline pretreatments. Alkaline pretreatments also solubilized most of the lignin.ConclusionsPretreatment pH exerted significant effects and factor interactions on the enzymatic glucose and xylose releases. Quite extreme pH values were necessary with mild thermal pretreatment strategies (T ≤ 140°C, time ≤ 10 min). Alkaline pretreatments generally induced higher enzymatic glucose and xylose release and did so at lower pretreatment temperatures than required with acidic pretreatments.

Discovery and industrial applications of lytic polysaccharide mono-oxygenases

The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine.

Lytic Polysaccharide Monooxygenases: The Microbial Power Tool for Lignocellulose Degradation

Lytic polysaccharide monooxygenases (LPMOs) are copper-enzymes that catalyze oxidative cleavage of glycosidic bonds. These enzymes are secreted by many microorganisms to initiate infection and degradation processes. In particular, the concept of fungal degradation of lignocellulose has been revised in the light of this recent finding. LPMOs require a source of electrons for activity, and both enzymatic and plant-derived sources have been identified. Importantly, light-induced electron delivery from light-harvesting pigments can efficiently drive LPMO activity. The possible implications of LPMOs in plant-symbiont and -pathogen interactions are discussed in the context of the very powerful oxidative capacity of these enzymes.

Cellulose is not just cellulose: a review of dislocations as reactive sites in the enzymatic hydrolysis of cellulose microfibrils

Most secondary plant cell walls contain irregular regions known as dislocations or slip planes. Under industrial biorefining conditions dislocations have recently been shown to play a key role during the initial phase of the enzymatic hydrolysis of cellulose in plant cell walls. In this review we chart previous publications that have discussed the structure of dislocations and their susceptibility to hydrolysis. The supramolecular structure of cellulose in dislocations is still unknown. However, it has been shown that cellulose microfibrils continue through dislocations, i.e. dislocations are not regions where free cellulose ends are more abundant than in the bulk cell wall. In more severe cases cracks between fibrils form at dislocations and it is possible that the increased accessibility that these cracks give is the reason why hydrolysis of cellulose starts at these locations. If acid or enzymatic hydrolysis of plant cell walls is carried out simultaneously with the application of shear stress, plant cells such as fibers or tracheids break at their dislocations. At present it is not known whether specific carbohydrate binding modules (CBMs) and/or cellulases preferentially access cellulose at dislocations. From the few studies published so far it seems that no special type of CBM is involved. In one case an endoglucanase was found to preferably bind to dislocations.