Ninety percent of lignocellulose-degrading fungi contain genes encoding lytic polysaccharide monooxygenases (LPMOs). These enzymes catalyze the initial oxidative cleavage of recalcitrant polysaccharides after activation by an electron donor. Understanding the source of electrons is fundamental to fungal physiology and will also help with the exploitation of LPMOs for biomass processing. Using genome data and biochemical methods, we characterized and compared different extracellular electron sources for LPMOs: cellobiose dehydrogenase, phenols procured from plant biomass or produced by fungi, and glucose-methanol-choline oxidoreductases that regenerate LPMO-reducing diphenols. Our data demonstrate that all three of these electron transfer systems are functional and that their relative importance during cellulose degradation depends on fungal lifestyle. The availability of extracellular electron donors is required to activate fungal oxidative attack on polysaccharides.
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O 2 − . Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.
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.
BackgroundCellobiose 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.ResultsWe 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.ConclusionsWhereas 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0653-5) contains supplementary material, which is available to authorized users.
a b s t r a c tA 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.
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