Cellobiose dehydrogenase in biofuel cells.
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ABSTRACT:
SUBMITTER: Scheiblbrandner S
PROVIDER: S-EPMC7613715 | biostudies-literature | 2022 Feb
REPOSITORIES: biostudies-literature
Publications
Cellobiose dehydrogenase in biofuel cells.
Scheiblbrandner Stefan S Csarman Florian F Ludwig Roland R
Current opinion in biotechnology 20210903
Enzymatic biofuel cells utilize oxidoreductases as highly specific and highly active electrocatalysts to convert a fuel and an oxidant even in complex biological matrices like hydrolysates or physiological fluids into electric energy. The hemoflavoenzyme cellobiose dehydrogenase is investigated as a versatile bioelectrocatalyst for the anode reaction of biofuel cells, because it is robust, converts a range of different carbohydrates, and can transfer electrons to the anode by direct electron tra ...[more]
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Project description:Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from cellobiose oxidation being shuttled from the CDH dehydrogenase domain to its cytochrome domain and then to the LPMO catalytic site. In this work, we investigate the interaction of four Neurospora crassa LPMOs and five CDH cytochrome domains from different species using computational methods. We used HADDOCK to perform protein-protein docking experiments on all 20 combinations and subsequently to select four complexes for extensive molecular dynamics simulations. The potential of mean force is computed for a rotation of the cytochrome domain relative to LPMO. We find that the LPMO loops are largely responsible for the preferred orientations of the cytochrome domains. This leads us to postulate a hybrid version of NcLPMO9F, with exchanged loops and predicted altered cytochrome binding preferences for this variant. Our work provides insight into the possible mechanisms of electron transfer between the two protein systems, in agreement with and complementary to previously published experimental data.
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Project description:A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.
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Project description:BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-β-D-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.
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Project description:Direct bioelectrocatalysis applied in biosensors, biofuel cells, and bioelectrosynthesis is based on an efficient electron transfer between enzymes and electrodes in the absence of redox mediators. Some oxidoreductases are capable of direct electron transfer (DET), while others achieve the enzyme to electrode electron transfer (ET) by employing an electron-transferring domain. Cellobiose dehydrogenase (CDH) is the most-studied multidomain bioelectrocatalyst and features a catalytic flavodehydrogenase domain and a mobile, electron-transferring cytochrome domain connected by a flexible linker. The ET to the physiological redox partner lytic polysaccharide monooxygenase or, ex vivo, electrodes depends on the flexibility of the electron transferring domain and its connecting linker, but the regulatory mechanism is little understood. Studying the linker sequences of currently characterized CDH classes we observed that the inner, mobile linker sequence is flanked by two outer linker regions that are in close contact with the adjacent domain. A function-based definition of the linker region in CDH is proposed and has been verified by rationally designed variants of Neurospora crassa CDH. The effect of linker length and its domain attachment on electron transfer rates has been determined by biochemical and electrochemical methods, while distances between the domains of CDH variants were computed. This study elucidates the regulatory mechanism of the interdomain linker on electron transfer by determining the minimum linker length, observing the effects of elongated linkers, and testing the covalent stabilization of a linker part to the flavodehydrogenase domain. The evolutionary guided, rational design of the interdomain linker provides a strategy to optimize electron transfer rates in multidomain enzymes and maximize their bioelectrocatalytic performance.
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Project description:Cellobiose dehydrogenase (CDH), a secreted flavocytochrome produced by a number of wood-degrading fungi, was detected in the culture supernatant of a biotechnologically important strain of Cerrena unicolor grown in a modified cellulose-based liquid medium. The enzyme was purified as two active fractions: CuCDH-FAD (flavin domain) (1.51-fold) with recovery of 8.35 % and CuCDH (flavo-heme enzyme) (21.21-fold) with recovery of 73.41 %. As CDH from other wood-rotting fungi, the intact form of cellobiose dehydrogenase of C. unicolor is a monomeric protein containing one flavin and one heme b with molecular mass 97 kDa and pI = 4.55. The enzyme is glycosylated (8.2 %) mainly with mannose and glucosamine residues. Moreover, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from the fungus C. unicolor were isolated, cloned, and characterized. The 2316-bp full-length cDNA of cdh1 encoded a mature CDH protein containing 771 amino acids preceded by a signal peptide consisting of 18 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.
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Project description:Covalent coupling between a surface exposed cysteine residue and maleimide groups was used to immobilize variants of Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) at multiwall carbon nanotube electrodes. By introducing individual cysteine residues at particular places on the surface of the flavodehydrogenase domain of the flavocytochrome we are able to immobilize the different variants in different orientations. Our results show that direct electron transfer (DET) occurs exclusively through the haem b cofactor and that the redox potential of the haem is unaffected by the orientation of the enzyme. Electron transfer between the haem and the electrode is fast in all cases and at high glucose concentrations the catalytic currents are limited by the rate of inter-domain electron transfer (IET) between the FAD and the haem. Using ferrocene carboxylic acid as a mediator we find that the total amount of immobilized enzyme is 4 to 5 times greater than the amount of enzyme that participates in DET. The role of IET in the overall DET catalysed oxidation was also demonstrated by the effects of changing Ca2+ concentration and by proteolytic cleavage of the cytochrome domain on the DET and MET currents.
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Project description:The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.
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Project description: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 H2 O2 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 H2 O2 -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 H2 O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals.
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