Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

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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.

Section: Resultssupporting

confidence: 79%

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

Tangthirasunun

Navarro

Garajová

et al. 2017

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“…8). Similarly, FgGaOx oxidized ␣-methyl galactopyranoside to corresponding galacturonic acid in H 2 18 O (see Fig. S5 in the supplemental material).…”

Section: Fig 4 Kinetic Behavior Ofmentioning

confidence: 92%

“…A positive-control reaction (to produce aldehyde) was performed by treating raffinose (dissolved in Milli-Q water instead of buffer) with galactose oxidase (0.5 U/mg substrate), HRP (1 U/mg substrate), and catalase (115 U/mg substrate) under the same conditions (16,37). High-dosage reactions were also conducted using H 2 18 O (97 atom % 18 O; Sigma-Aldrich) as a solvent. 1 H NMR spectra were obtained using a Varian Innova 500 spectrometer (Varian NMR Systems, Palo Alto, CA) operating at 500 MHz.…”

Section: Methodsmentioning

confidence: 99%

“…The mechanism of formation of the acid was the oxidation of hydrate form of the aldehyde product, as shown by performing oxidation in H 2 18 O. The FgGaOx-catalyzed reaction conditions could be controlled to avoid the formation of the acid (37), and most probably such reaction conditions could also be found for the CgRaOx-catalyzed reaction.…”

Section: Characterization Of a Novel Raffinose Oxidasementioning

confidence: 99%

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A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Andberg

Mollerup

Parikka

et al. 2017

We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.Ϫ) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on D-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1 H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O 2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.KEYWORDS CAZy AA5, galactose oxidase, nuclear magnetic resonance, NMR, carbohydrate, EC 1.1.3.Ϫ E nzymes that cleave or form oligo-and polysaccharides are classified in the sequence-based carbohydrate-active enzyme database (CAZy [http://www.cazy .org]). The sequences are linked to information about functionality (e.g., substrate specificity) and three-dimensional structure. The CAZy database was recently expanded to account for redox enzymes, leading to the auxiliary activities (AA) group. Currently, 13 AA families exist, comprising lignolytic oxidoreductases (families AA1, -2, -4, and -6), lytic polysaccharide monooxygenases (families AA9, -10, -11, and -13), carbohydrate

“…Since their discovery, LPMOs received much attention and are currently considered one of the key enzymes in fungal cellulose degradation (8). LPMOs employ molecular oxygen and an external electron donor (5, 9–12) to carry out oxidative cleavage of the β-1,4-glucosidic bonds in cellulose. Some LPMOs exclusively oxidize C-1, others exclusively oxidize C-4, whereas a third type of LPMOs yields a mixture of C-1- and C-4-oxidized products.…”

Section: Introductionmentioning

confidence: 99%

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

Kojima

Várnai

Ishida

et al. 2016

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.