Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Nicht verfügbarMultigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucosemethanolcholine (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.(VLID)192910

Cellulose degrading oxidoreductases

Holz ist das größte Kohlenhydratvorkommen unseres Planeten und wird mittelfristig eine wichtige Rolle in der weltweiten Energieversorgung spielen. Holz (bzw. Lignozellulose) kann mittels spezifischer Enzyme, welche von holzzersetzenden Pilzen produziert werden, in seine molekularen Zuckerkomponenten zerlegt werden. Diese Arbeit ergründet die Funktion des Flavocytochroms Cellobiosedehydrogenase (CDH) in diesem lignozellulolytischen Prozess und studiert dessen Interaktion mit dem kupferhältigen Enzym lytische Polysaccharid-Monoxygenase (LPMO). CDHs bestehen aus einem Häm b hältigen Fragment (CYT), welches mit einer katalytischen Flavodehydrogenase (DH) verbunden ist. Biochemische Analysen in Kombination mit den ersten Kristallstrukturen von CDHs zeigen eine sehr dynamische Interaktion zwischen diesen Enzymen. Dabei aktiviert (reduziert) CDH die LPMO mittels der flexiblen CYT-Domäne, welche als mobiler Elektronen-Mediator fungiert. Pilze, denen CDH Aktivität fehlt, können auch auf andere Oxidoreduktasen zurückgreifen, welche die LPMO indirekt über phenolische Holzkomponenten, die als Redoxmediatoren wirken, aktivieren. Sind solche Phenole nicht verfügbar, können einige Pilze diese Stoffe auch selbst sekretieren, um die LPMO-Aktivät aufrecht zu erhalten. Die Verwendung mehrerer Elektronenquellen erlaubt es Pilzen, sich an unterschiedliche Umwelt- und Wachstumsbedingungen anzupassen und ist somit ein wichtiger Faktor in der Pilzphysiologie. Die katalytische Diversität der untersuchten LPMOs und CDHs, die in einer Reihe von Publikationen in dieser Dissertation dokumentiert ist, verdeutlicht zudem die Wichtigkeit der Suche nach neuen Enzymvarianten und deren biochemischer Analyse. Ein grundsätzliches Verständnis für die Aktivität, Interaktion und die vielfältigen katalytischen Eigenschaften dieser Enzyme ist entscheidend für deren Anwendung in Bioraffinerien, die aus dem Rohstoff Holz energiereiche Kohlenwasserstoffe und Chemikalien zu generieren suchen.Wood represents the largest terrestrial carbohydrate resource and will play a crucial role as renewable feedstock in the future energy supply. Degradation into its sugar constituents can be achieved by highly specific enzymes produced by wood-rotting fungi. This thesis explores the role of cellobiose dehydrogenase (CDH), a fungal flavocytochrome, in lignocellulolytic processes and studies its interaction with the copper-dependent lytic polysaccharide monooxygenase (LPMO). CDH has a two-domain architecture comprising a heme b-containing moiety (CYT) connected to a catalytic flavodehydrogenase (DH) domain. Biochemical analyses in combination with the first crystal structures of full-length CDHs demonstrate a dynamic interaction between these enzymes, in which CDH reductively activates LPMO via its mobile CYT fragment. Fungi lacking CDH activity may also rely on other oxidative enzymes, which are able to activate LPMO indirectly via wood-derived phenolic redox-mediators. Depending on the availability of such phenols, some fungi are able to secrete these compounds themselves in order to maintain LPMO activity. The utilization of several electron sources allows fungi to adapt to different growth conditions or habitats and therefore is an elementary aspect in fungal physiology. The catalytic diversity of the herein studied LPMOs and CDHs, which is documented in a series of publications within this thesis, underscores the importance of identifying and investigating new enzyme variants. A general understanding of the activity, interaction and catalytic features of these fascinating redox enzymes is crucial for their application in biorefineries to efficiently transform plant biomass into hydrocarbons and commodity chemicals.submitted by Daniel KracherZusammenfassung in deutscher SpracheUniversität für Bodenkultur Wien, Dissertation, 2016OeBB(VLID)193035

Electron Transfer of Cellobiose Dehydrogenase in Polyethyleneimine Films

Abstract Cellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications

Inter-domain electron transfer in cellobiose dehydrogenase: modulation by pH and divalent cations

The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH(2) in the catalytic flavodehydrogenase domain of CDH to haemb in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of >3mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haemb propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect

A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides

193817, 203402, 214613, 216162 and 217708,publishedVersio

Engineering an enzymatic regeneration system for NAD(P)H oxidation

AbstractA recently proposed coenzyme regeneration system employing laccase and a number of various redox mediators for the oxidation of NAD(P)H was studied in detail by kinetic characterization of individual reaction steps. Reaction engineering by modeling was used to optimize the employed enzyme, coenzyme as well as redox mediator concentrations. Glucose dehydrogenase from Bacillus sp. served as a convenient model of synthetic enzymes that depend either on NAD+ or NADP+. The suitability of laccase from Trametes pubescens in combination with acetosyringone or syringaldazine as redox mediator was tested for the regeneration (oxidation) of both coenzymes. In a first step, pH profiles and catalytic constants of laccase for the redox mediators were determined. Then, second-order rate constants for the oxidation of NAD(P)H by the redox mediators were measured. In a third step, the rate equation for the entire enzymatic process was derived and used to build a MATLAB model. After verifying the agreement of predicted vs. experimental data, the model was used to calculate different scenarios employing varying concentrations of regeneration system components. The modeled processes were experimentally tested and the results compared to the predictions. It was found that the regeneration of NADH to its oxidized form was performed very efficiently, but that an excess of laccase activity leads to a high concentration of the oxidized form of the redox mediator – a phenoxy radical – which initiates coupling (dimerization or polymerization) and enzyme deactivation

Inhibition of the Peroxygenase Lytic Polysaccharide Monooxygenase by Carboxylic Acids and Amino Acids

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions on the activity and stability of LPMO during catalysis. Turbidimetric and photometric assays were used to identify LPMO inhibitors and measure their inhibitory effect. Selected inhibitors were employed to study LPMO activity and stability during cellulose depolymerization by HPLC and turbidimetry. It was found that the fungal metabolic products oxalic acid and citric acid strongly reduce LPMO activity, but also protect the enzyme from deactivation. QM calculations showed that the copper atom in the catalytic site could be ligated by bi- or tridentate chelating compounds, which replace two water molecules. MD simulations and QM calculations show that the most likely inhibition pattern is the competition between the inhibitor and reducing agent in the oxidized Cu(II) state. A correlation between the complexation energy and the IC50 values demonstrates that small, bidentate molecules interact strongest with the catalytic site copper and could be used by the fungus as physiological effectors to regulate LPMO activity