Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation

Abstract

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.

Keywords: CBM33; GH61; biorefinery; metallo enzymes.

Fig. 1.

Glycan microarray screening of NcLPMO9C activity on various polysaccharide substrates. Activities were detected by the loss or reduction of polysaccharide-borne epitopes recognized by substrate-specific monoclonal antibodies (mAb). (A) Heatmap showing degradation of the substrates listed above the panel following treatment with NcLPMO9C at different concentrations (listed left), in the presence (+) or absence (−) of ascorbic acid. Control means no enzyme treatment. The mAbs used to recognize the substrates are in parentheses, top row. The heatmap shows relative mean spot signal intensities; the lower the signal when comparing controls and NcLPMO9C treated samples, the greater the degree of degradation. (B) Fold-change heatmap of the data shown in A. This heatmap shows the degree of change in mAb binding resulting from NcLPMO9C treatment of the substrates. A cutoff of 5 was imposed before the fold changes were calculated. (C) Images of the arrays used to produce the heatmaps, showing consistent spot morphologies and depleted signals caused by NcLPMO9C. * denotes that final concentration of xyloglucan was 1 mg/mL, whereas the other substrates were 0.1 mg/mL.

Fig. 2.

MALDI-ToF MS analysis of product profiles. The spectra show products generated from tamarind xyloglucan (A; see Inset regarding nomenclature; blue, glucose; orange, xylose; yellow, galactose), konjac glucomannan (B), lichenan and mixed linked glucan (MLG) from barley (C) treated with either endo-glucanase (magenta) or LPMO (black, turquoise for MLG). Product profiles upon endo-glucanase treatment of lichenan and MLG were essentially the same and therefore only one of the spectra is shown. Brackets in B indicate product clusters of same DP, indicated by the number. In the main spectra, only sodium adducts are labeled, whereas the inserts also show potassium adducts (marked *) and various forms of oxidized species where both the keto-group formed upon C4 oxidation (−2 Da) and its gemdiol form (marked #, i.e., addition of H₂O, +18 Da) appear. Abbreviations: G, X. and L, see A Inset; Glc, glucose (+ 162 Da); Hex, hexose (+ 162 Da); Ac, acetyl group (+ 42 Da); ox, oxidized (−2 Da if keto form). Analysis of control reactions, without enzyme addition, showed no signals related to carbohydrates.

Fig. 3.

Analysis of products generated from XG14^OH. (A) ESI-MS spectra highlighting Na-adducts of products generated by NcLPMO9C (black) or AfCel12A (magenta). ESI-MS spectra show primarily the gemdiol form (marked #) of oxidized products. (B) MALDI-ToF-MS spectra highlighting Na-adducts of XG14^OH (blue) and of products generated from XG14^OH by NcLPMO9C (black) or AfCel12A (magenta). MALDI-ToF-MS spectra show primarily the keto-form of oxidized products, which in the case of a reduced substrate has the same m/z as native products (keto group gives m/z -2, and reduction gives m/z +2). Note that panel B shows that XG14^OH (XXXGXXXG^OH) is contaminated with other species containing one or more additional hexoses, probably galactoses coupled to one or more of the X units as this is a very common moiety in xyloglucan from tamarind (hence annotation as L in the figure). Some products derived from these contaminations are annotated in the mass spectra. (C) MS2 fragmentation of an m/z 1,249 species generated upon lithium doping of the product mixture shown in panel A (m/z 1,249 corresponds to the Li-adduct of the m/z 1,265 species in A). Gemdiol-fragments readily lose a water molecule thus occurring in the spectrum as B or C −18 Da (fragmentation nomenclature as in ref. 30); this has been observed previously for gemdiol products (12). The substrate, XG14^OH, and possible products of m/z 1,249 are shown as cartoons according to the nomenclature of (31): blue circle, glucose; orange star, xylose; yellow circle, galactose. Parenthesis surrounding galactosyl-units denote that the position of these units may vary. “Ox” denotes the position of the oxidation. “Red” denotes the position of reduction. Note that dominating fragmentation reactions lead to removal of substitutions from the glucan backbone, explaining why several oligo-G products are detected.

Fig. 4.

MALDI-ToF spectra of products generated from natural substrates. Arabidopsis (A) or tomato stem (B) cell wall material was incubated with either NcLPMO9C (black line) or AfCel12A (magenta line) and product mixtures were analyzed. Peaks were assigned by combining the mass information with knowledge on the substrate and the preferences of the enzymes in question; note that these assignments to some extent are arbitrary, because the exact positions of modifications/substitutions cannot always be inferred. Also note that additional sugars and modifications occur in these natural substrates: F, fucosylation on an l-unit (+146 Da); S, arabinosyl substitution on an X-unit (+132 Da). Acetylations (+42 Da) are also observed and marked by underlined G, F, or S units. All assigned masses are sodium adducts, except when indicated otherwise. Potassium adducts are marked by an asterisk. §, background signal. Control reactions showed that ascorbic acid alone did not release any products and did not change the product profile generated by the endo-glucanase.