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

Abstract

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.

FIG 1

Position of G. trabeum LPMOs in the phylogenetic tree of LPMO9s. The phylogenetic tree was created from catalytic domains of LPMO9s by using the neighbor-joining method in ClustalX (version 2.1). Several LPMOs are labeled, and their cleavage specificities are indicated (C-1, C-4, or both [C-1/C-4]; N. D., not determined). For all labeled non-GtLPMOs, activity on cellulose has been demonstrated. An asterisk indicates that additional activity on xyloglucan has been detected. The functional data for GtLPMO9A-2 stem from the present report; other functional data come from references , , , , and 65).

FIG 2

Multiple-sequence alignment of the catalytic domains of selected LPMO9s. Fully conserved residues are printed in white on a black background. Active-site histidines (black-filled triangles) and a tyrosine (gray-filled triangle) involved in copper coordination are indicated. The labeled bars over the sequences indicate known variable regions in LPMO9s (15, 51). Note that GtLPMO9A-2 and GtLPMO9D have C-terminal extensions; for more information, see Fig. S2 to S4 in the supplemental material. This alignment was generated using MAFFT, version 7.295 (39), available at the European Bioinformatics Institute website (https://www.ebi.ac.uk/Tools/msa/mafft/).

FIG 3

Products generated by GtLPMO9A-2 or NcLPMO9C on PASC. (A) HPAEC-PAD chromatograms showing cello-oligosaccharides released by GtLPMO9A-2 (blue) and NcLPMO9C (red) from PASC. Peaks were assigned based on previous assignments by Isaksen et al. (50); native cello-oligosaccharides are labeled as Glc_n, where n is the degree of polymerization (DP). Note that it has recently been shown that C-4-oxidized products are unstable under these chromatographic conditions and that the peaks labeled C-4 are, in fact, diagnostic degradation products (34). The fractions of native products are high because C-4-oxidized products tend to lose the oxidized monosugar under these chromatographic conditions. (B) MALDI-TOF spectrum of cello-oligosaccharides released by GtLPMO9A-2 from PASC, where the inset shows details for the heptamer ion cluster (sodium adducts only). Possible products in these clusters are the native Glc₇ (m/z 1,175.8), the C-1-oxidized lactone, or C-4-oxidized ketoaldose (anhydrated species, m/z 1,173.8), the C-1-oxidized aldonic acid or C-4-oxidized gemdiol (hydrated species, m/z 1,191.8), and the sodium adduct of the aldonic acid sodium salt (m/z 1,213.8). The double-oxidized species corresponds to m/z 1,171.8 (anhydrated form, lactone-ketoaldose species), m/z 1,189.8 (hydrated form), and m/z 1,211.8 (the sodium salt of the sodium adduct). The presence of sodium salts is diagnostic for C-1 oxidation, since only this oxidation yields an aldonic acid. The strong signals for dehydrated oxidized species in MALDI-TOF are diagnostic for C-4 oxidation. The 4-keto to gemdiol equilibrium is less skewed toward the hydrated form than the lactone-aldonic acid equilibrium, and, besides, C-4-oxidized products are more efficiently dehydrated during spotting on MALDI sample plates (see Forsberg et al. [66] for further discussion). Experiments with Avicel yielded similar product patterns (data not shown).

FIG 4

Assessing LPMO activity on hemicelluloses with dynamic viscosity experiments. GtLPMO9A-2 (a, c, e, and g) or NcLPMO9C (b, d, f, and h) was incubated with 0.15% (wt/vol) xyloglucan (a and b), 0.05% (wt/vol) glucomannan (c and d), 0.2% (wt/vol) arabinoxylan (e and f), and 0.5% (wt/vol) carboxymethyl cellulose (g and h) in the presence (red lines) or absence (green lines) of DTT as a reducing agent. Reactions with only DTT and no LPMO are shown by blue lines.

FIG 5

HPAEC-PAD analysis of reaction products generated from xyloglucan (XG) by GtLPMO9A-2 and NcLPMO9C in the dynamic viscosity experiments. Samples were taken after 16 h of incubation, and the chromatograms show the product profiles before (A) and after (B) a subsequent treatment with AfCel12A. Green lines, XG7 standard (XXXG); gray lines, XG-oligomer standard (this is a mixture of shorter XG oligomers with DP in the range of 14 to 27); black lines, XG incubated with DTT only; red lines, products generated from XG with NcLPMO9C in the presence (dark red) or absence (light red) of DTT; blue lines, products generated from XG with GtLPMO9A-2 in the presence (dark blue) or absence (light blue) of DTT. Gray dashed lines indicate oligosaccharides, the concentrations of which were lower as a result of LPMO activity. The reaction conditions are specified in Materials and Methods. Note that Fig. 7 provides an even clearer example of the difference between NcLPMO9C and GtLPMO9A-2 with respect to the activity on xyloglucan.

FIG 6

MALDI-TOF MS analysis of the reaction products generated by GtLPMO9A-2 (A) and NcLPMO9C (B) during the dynamic viscosity experiments with tamarind xyloglucan (XG) in the presence of reducing agent (DTT). The samples analyzed were the endpoint samples (16 h). The reaction conditions are specified in Materials and Methods and were similar to the conditions used for generating Fig. 5. GtLPMO9A-2 generated a wide range of oligosaccharides with all possible combination of hexose (Hex) and pentose (Pen) units, whereas NcLPMO9C generated clusters of oligosaccharides with the number of pentose residues being a multiple of three. In both spectra, oxidized species (−2, compared with the weight of native species) are marked with #; species with masses indicating arabinosylation are labeled green (see the text). Note that most labeled peaks in fact are a cluster of signals; see Fig. S10 in the supplemental material and text. All labeled species are Na⁺ adducts. No species were detected below m/z 1,000.

FIG 7

HPAEC-PAD analysis of the reaction products generated by GtLPMO9A-2 (A) or NcLPMO9C (B) on xyloglucan-coated PASC. Green lines, native cello-oligosaccharides with DP 2 to 6; yellow lines, XG7 standard (XXXG); gray lines, XG-oligomer standard with DP 14 to 27; brown lines, mixture of PASC and XG incubated with LPMO only; black lines, mixture of PASC and XG incubated with ascorbic acid (ASC) only; blue lines, products generated from PASC with LPMO in the presence of ASC; red lines, products generated from XG with LPMO in the presence of ASC; purple lines, products generated from xyloglucan-coated PASC with LPMO in the presence of ASC. The reaction conditions are specified in Materials and Methods. Note that the chromatographic conditions were similar to those used for the experiment whose results are shown in Fig. 5 but different from those used for the experiment whose results are shown in Fig. 3. This explains why the retention times of cello-oligomers differ between the data in Fig. 3 and 5.