Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

Tan, T. K.; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hållberg, Bengt; Ludwig, Roland; Divne, Christina

doi:10.1038/ncomms8542

Cited by 213 publications

(340 citation statements)

References 62 publications

(87 reference statements)

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“…All substrates had a substantial effect on the chemical shifts of residues His1, Ala80, His83 and His155, whereas the effect on the chemical shifts of other amino acids varied according to the substrates used. A large surface loop [also known as the LC loop (31)] showing considerable variation among LPMOs but also containing a highly conserved tyrosine, Tyr204, which has been suggested to contribute to cellulose binding (13,31), was generally little affected by substrate binding and was more affected by the binding of XG 14 and polyXG than by the binding of Glc 6 (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…LPMOs are abundantly present in biomass-degrading microbes and make use of molecular oxygen and an external electron donor to cleave polysaccharides through hydroxylation of one of the carbons in the scissile glycosidic bond (4,5,(9)(10)(11)(12)(13). LPMOs can accept electrons from cellobiose dehydrogenase (CDH) (3,14,15) or a variety of small molecule reducing agents such as ascorbate and gallic acid (4,5) as well as lignin-derived redox mediators (16). Each LPMO reaction cycle is postulated to consume two electrons (3,5,6).…”

mentioning

confidence: 99%

“…According to the postulated LPMO mechanisms, CDH has to deliver two electrons to the LPMO during each reaction cycle (6,10,29). Docking studies have suggested that surface residues close to the copper site interact with the cytochrome domain of CDH during electron transfer (ET) (14). An alternative CDH docking site that would not be blocked by substrate binding has also been suggested (13).…”

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confidence: 99%

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Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Courtade

Wimmer

Røhr

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Courtade

Wimmer

Røhr

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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134

149

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“…LPMOs use an oxidative mechanism to introduce chain breaks into polysaccharides thereby augmenting the activity of classical glycoside hydrolases, as shown by Vaaje Kolstad et al 13 in their breakthrough study of CBP21 (chitin-binding protein 21) from Serratia marcescens. Using a copper co-factor together with an electron source which can be a small-molecule reducing agent, such as ascorbate 14 , or a protein partner such as cellobiose dehydrogenase (CDH) in fungi [15][16][17] , LPMOs introduce a single atom from O 2 at either the C1 or C4 position of the sugar ring, destabilising the glycoside linkage resulting in chain cleavage 2,[14][15][16]18 . The strength of the scissile C-H bond which has been estimated to be ca 95 kcal/mol, indicative of the powerful oxidative nature of LPMOs.…”

Section: Introductionmentioning

confidence: 99%

Activity, stability and 3-D structure of the Cu(ii) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens

et al. 2016

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The enzymatic deconstruction of recalcitrant polysaccharide biomass is central to the conversion of these substrates for societal benefit, such as in biofuels. Traditional models for enzyme-catalysed polysaccharide degradation involved the synergistic action of endo-, exo-and processive glycoside hydrolases working in concert to hydrolyse the substrate. More recently this model has been succeeded by one featuring a newly discovered class of mononuclear copper enzymes: lytic polysaccharide monooxygenases (LPMOs; classified as Auxiliary Activity (AA) enzymes in the CAZy classification). In 2013, the structure of an LPMO from Bacillus amyloliquefaciens, BaAA10, was solved with the Cu centre photoreduced to Cu(I) in the X-ray beam. Here we present the catalytic activity of BaAA10. We show that it is a chitin-active LPMO, active on both α and β chitin, with the Cu(II) binding with low nM KD, and the substrate greatly increasing the thermal stability of the enzyme. A spiral data collection strategy has been used to facilitate access to the previously unobservable Cu(II) state of the active centre, revealing a coordination geometry around the copper which is distorted from axial symmetry, consistent with the previous findings from EPR spectroscopy.

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“…The electrons are rapidly transferred from the flavin to the heme domain of CDH via the heme propionate group (24)(25)(26). Following heme reduction, the cytochrome domain of CDH reduces the copper in the active site of the LPMO to initiate oxidative cellulose breakdown (27).…”

mentioning

confidence: 99%

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

Tangthirasunun

Navarro

Garajová

et al. 2017

Appl Environ Microbiol

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Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi.IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

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Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

Cited by 213 publications

References 62 publications

Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Activity, stability and 3-D structure of the Cu(ii) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

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