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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. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H2O2 rather than O2 is the cosubstrate of the LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H2O2-based mechanism for plant cell wall degradation. This study reports the localized and time-resolved determination of LPMO activity on poplar wood cell walls by measuring the H2O2 concentration in their vicinity with a piezo-controlled H2O2 microsensor. The investigated Neurospora crassa LPMO binds to the inner cell wall layer and consumes enzymatically generated H2O2. The results point towards a high catalytic efficiency of LPMO at a low H2O2 concentration that auxiliary oxidoreductases in fungal secretomes can easily generate. Measurements with a glucose microbiosensor additionally demonstrate that LPMO promotes cellobiohydrolase activity on wood cell walls and plays a synergistic role in the fungal extracellular catabolism and in industrial biomass degradation. It is important to understand the enzymatic degradation of wood biomass by lytic polysaccharide monooxygenase, however, disagreements about the co-substrate exist. Here, the authors use piezo-controlled hydrogen peroxide micro-sensors to demonstrate that even low levels of hydrogen peroxide support the enzymatic degradation of wood cell walls. [ABSTRACT FROM AUTHOR]

The textile industry is one of the largest water-polluting industries in the world. Due to an increased application of chromophores and a more frequent presence in wastewaters, the need for an ecologically favorable dye degradation process emerged. To predict the decolorization rate of textile dyes with Lytic polysaccharide monooxygenase (LPMO), we developed, validated, and utilized the molecular descriptor structural causality model (SCM) based on the decision tree algorithm (DTM). Combining mathematical models and theories with decolorization experiments, we have elucidated the most important molecular properties of the dyes and confirm the accuracy of SCM model results. Besides the potential utilization of the developed model in the treatment of textile dye-containing wastewater, the model is a good base for the prediction of the molecular properties of the molecule. This is important for selecting chromophores as the reagents in determining LPMO activities. Dyes with azo- or triarylmethane groups are good candidates for colorimetric LPMO assays and the determination of LPMO activity. An adequate methodology for the LPMO activity determination is an important step in the characterization of LPMO properties. Therefore, the SCM/DTM model validated with the 59 dyes molecules is a powerful tool in the selection of adequate chromophores as reagents in the LPMO activity determination and it could reduce experimentation in the screening experiments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2O2‐driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped‐flow spectroscopy, alongside electron paramagnetic resonance and UV‐Vis spectroscopy, we reveal how H2O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions. [ABSTRACT FROM AUTHOR]

Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. Results: We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. Conclusions: We found for all systems that the supply of H2O2 limited LPMO's cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods. [ABSTRACT FROM AUTHOR]

The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: an engineered fungal cellobiose dehydrogenase (CDH) with increased oxidase activity. The engineered CDH was about 30 times more efficient in driving the LPMO reaction due to its 27 time increased production of H2O2 acting as a cosubstrate for LPMO. Transient kinetic measurements confirmed that intra‐ and intermolecular electron transfer rates of the engineered CDH were similar to the wild‐type CDH, meaning that the mutations had not compromised CDH's role as an electron donor. These results support the notion of H2O2‐driven LPMO activity and shed new light on the role of CDH in activating LPMOs. Importantly, the results also demonstrate that the use of the engineered CDH results in fast and steady LPMO reactions with CDH‐generated H2O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals. [ABSTRACT FROM AUTHOR]

This review aims to present current knowledge of the fungi involved in lignocellulose degradation with an overview of the various classes of lignocellulose‐acting enzymes engaged in the pretreatment and saccharification step. Fungi have numerous applications and biotechnological potential for various industries including chemicals, fuel, pulp, and paper. The capability of fungi to degrade lignocellulose containing raw materials is due to their highly effective enzymatic system. Along with the hydrolytic enzymes consisting of cellulases and hemicellulases, responsible for polysaccharide degradation, they have a unique nonenzymatic oxidative system which together with ligninolytic enzymes is responsible for lignin modification and degradation. An overview of the enzymes classification is given by the Carbohydrate‐Active enZymes (CAZy) database as the major database for the identification of the lignocellulolytic enzymes by their amino acid sequence similarity. Finally, the recently discovered novel class of recalcitrant polysaccharide degraders‐lytic polysaccharide monooxygenases (LPMOs) are presented, because of these enzymes importance in the cellulose degradation process. [ABSTRACT FROM AUTHOR]

This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103- to 104-fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. [ABSTRACT FROM AUTHOR]

Multigenicity 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 glucose-methanol-choline (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. [ABSTRACT FROM AUTHOR]

BACKGROUND Sugar beet pulp ( SBP) is a promising feedstock for the production of 2nd generation biofuels, but efficient enzymatic hydrolysis remains a key challenge; therefore, new process designs and/or bioreactor designs are crucial to overcome this hurdle. In this regard, horizontal rotating tubular bioreactors ( HRTB) offer the advantage of high substrate loadings while minimizing the space and energy demand compared with conventional stirred tank reactors. Here, a statistical approach is used to optimize the hydrolysis of sugar beet pulp in laboratory experiments, and it is shown that such a process can be implemented in a HRTB. RESULTS Using the design of experiments ( DOE) method, the reaction conditions of four commercial enzyme mixtures (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase) was optimized for the degradation of SBP in small-scale experiments. Using Ultrazym AFP-L as the most efficient mixture, a 10 L scale conversion was performed in a HRTB. At a substrate loading of 135 g L−1 and optimized conversion parameters (enzyme load, pH and rotating speed of the reactor), 0.525 W dm−3 were needed to achieve solubilisation of 30% of the total mass of initial SBP after 24 h. CONCLUSION DOE was found to be an easy-to-apply method that allowed optimizing the conditions for enzymatic hydrolysis of SBP, resulting in a higher sugar yield. The results could be transferred to an HRTB, which is a suitable system for enzymatic conversion and efficient saccharification of semi-solid or solid substrates with relatively low energy consumption. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background: Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity. Results: We compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability. Conclusions: Whereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate-active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper-dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum ( MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar. [ABSTRACT FROM AUTHOR]

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Background: Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition. Results: Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L-1 for PsLPMOA and >260 mg L-1 for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stability and activity in the presence of sea salt. Both PsLPMOs were activated by a cellobiose dehydrogenase (CDH) from Neurospora crassa, with an apparent optimum of interaction at pH 5.5. Investigation into their regioselective mode of action revealed that PsLPMOA released C1- and C4-oxidized cello-oligosaccharide products, while PsLPMOB released only C4-oxidized products. PsLPMOA was found to cleave polymeric cellulose in the presence of up to 6 % sea salt, which emphasizes the use of sea water in the industrial saccharification process with improved ecological footprints. Conclusions: Two new LPMOs from the mangrove fungus Pestalotiopsis sp. NCi6 were found to be fully reactive against cellulose. The combined hydrolytic activities of these salt-responsive LPMOs could therefore facilitate the saccharification process using sea water as a reaction medium for large-scale biorefineries. [ABSTRACT FROM AUTHOR]

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 FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b 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 > 3 m m, 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 haem b 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. [ABSTRACT FROM AUTHOR]

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP- d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP- d-glucose is converted via UDP-4-keto- d-glucose to UDP- d-glucose-5,6- ene, the structure of which was determined by H and C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP- d-glucose and NAD could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP- d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested. [ABSTRACT FROM AUTHOR]

Polymer-multiwalled carbon nanotube (MWCNT) nanohybrids, which differ in surface charge have been synthesized to study the bioelectrocatalysis of adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete sordida on gold electrodes. To obtain negatively charged nanohybrids, poly(3-amino-4-methoxybenzoic acid-co-aniline) (P(AMB-A)) was covalently linked to the surface of MWCNTs while modification with p-phenylenediamine (PDA) converted the COOH-groups to positively charged amino groups. Fourier transform infrared spectroscopy (FTIR) measurements verified the p-phenylenediamine (PDA) modification of the polymer-CNT nanohybrids. The positively charged nanohybrid MWCNT-P(AMB-A)-PDA promoted direct electron transfer (DET) of CDH to the electrode and bioelectrocatalysis of lactose was observed. Amperometric measurements gave an electrochemical response with KMapp = 8.89 mM and a current density of 410 nA/cm2 (15 mM lactose). The catalytic response was tested at pH 3.5 and 4.5. Interference by ascorbic acid was not observed. The study proves that DET between the MWCNT-P(AMB-A)-PDA nanohybrids and CDH is efficient and allows the sensorial detection of lactose. [ABSTRACT FROM AUTHOR]

Background: Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Results: Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. Conclusions: P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components. [ABSTRACT FROM AUTHOR]

The long road from emerging biotechnologies to commercial "green" biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or "saccharification" requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test-Learn methodologies. These rapid automated pathway building infrastructures could be adapted for addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially viable levels. [ABSTRACT FROM AUTHOR]

The pentose phosphate pathway (PPP) is a route that can work in parallel to glycolysis in glucose degradation in most living cells. It has a unidirectional oxidative part with glucose-6-phosphate dehydrogenase as a key enzyme generating NADPH, and a non-oxidative part involving the reversible transketolase and transaldolase reactions, which interchange PPP metabolites with glycolysis. While the oxidative branch is vital to cope with oxidative stress, the non-oxidative branch provides precursors for the synthesis of nucleic, fatty and aromatic amino acids. For glucose catabolism in the baker's yeast Saccharomyces cerevisiae, where its components were first discovered and extensively studied, the PPP plays only a minor role. In contrast, PPP and glycolysis contribute almost equally to glucose degradation in other yeasts. We here summarize the data available for the PPP enzymes focusing on S. cerevisiae and Kluyveromyces lactis, and describe the phenotypes of gene deletions and the benefits of their overproduction and modification. Reference to other yeasts and to the importance of the PPP in their biotechnological and medical applications is briefly being included. We propose future studies on the PPP in K. lactis to be of special interest for basic science and as a host for the expression of human disease genes. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurospora crassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis. [ABSTRACT FROM AUTHOR]