Different sources of hemicelluloses were incubated with PaLPMO9H under ascorbate conditions and their reaction products were characterized by mass spectrometry. Four substrates were considered: lichenan, MLG, XyG and GM. Cellulose was also included in the study as a reference substrate. The activity of PaLPMO9H products on cellulose has been described in detail and was shown to generate singly and doubly oxidized products at the C1 and/or C4 positions. This results in the formation of gem-diols/ketones at the newly formed “non-reducing” end or aldonic acids at the newly formed “reducing” end [15].
PaLPMO9H demonstrates broad specificity toward β-(1 → 4)-linked glucans
As shown by the mass spectra in Fig. 1b–e, the incubation of PaLPMO9H with the four hemicellulosic substrates produced some oligosaccharides having degrees of polymerization (DPs) ranging from 2 to 12, confirming the depolymerizing activity of the enzyme on all of these structures. The most abundant peaks ranged between DP2 and DP4 for lichenan, MLG, and GM. In the case of XyG, DP6 and DP7 were detected with the highest intensity. In addition, the presence of some species with a mass shift of −2 (ketone or lactone) and +16 (gem-diol or aldonic acid) Da relative to the non-modified oligosaccharide suggested an oxidative cleavage activity, in agreement with the previous observations on cellulose [15]. The profiles of the reaction products were quite similar for all hemicellulosic substrates (Fig. 1, right panel). The non-modified species were always the most intense, regardless of the DP, while the intensity of the −2 Da species was either higher or similar to that of +16 Da species. The situation was slightly different when PaLPMO9H was incubated with cellulose (Fig. 1a). The species at −2 and +16 Da were the most intense ions. One additional species of low intensity was detected with a mass shift of +32 Da (m/z 721.20) from the related non-modified DP. This species has been described as a doubly oxidized product, with one gem-diol on the non-reducing end (C4), and another oxidation either at the reducing end or at the non-reducing end [15].
These results unambiguously confirmed that PaLPMO9H enzyme exhibits a broad specificity toward hemicelluloses compared to other AA9 LPMOs. Subtle differences were observed with respect to the action on cellulose, among which the apparent absence of double-oxidation. In-depth characterization of the reaction products was further carried out by tandem MS to explore the regioselectivity of the oxidative cleavage.
PaLPMO9H oxidatively cleaves β-(1 → 4)- and β-(1 → 4; 1 → 3)-linked glucosidic substrates
Lichenan and MLG are each composed of a linear chain of β-d-glucosyl residues linked by (1 → 4) and (1 → 3) linkages. The proportion and distribution of these β-(1 → 4) and β-(1 → 3) linkages vary between these two substrates; the average ratio of β-(1 → 3):β-(1 → 4) bonds is roughly 1:3 and 1:2 for MLG and lichenan, respectively. To differentiate possible linkage isomers among the reaction products, ion mobility was used in combination with tandem MS. Ion mobility separates species based on their gas-phase conformation prior to their mass measurement. Figure 2 shows the mobilograms recorded for the non-modified species of DP4 obtained for lichenan, MLG and cellulose. As cellulose is exclusively composed of β-(1 → 4) linkages, its degradation was expected to produce a single DP4 species that could be used as a reference. Figure 2 shows that a major peak at a drift time (dt) of 76 bins was detected for the natriated non-oxidized DP4 ion (m/z 689.2) arising from cellulose. In contrast, mobility traces indicated the clear presence of at least two gas-phase isoforms for the DP4 released from lichenan and MLG. One of those (dt = 76 bins) corresponded to a DP4 species containing three consecutive β-(1 → 4) linkages (cellotetraose). The second one (dt = 83 bins) was assumed to contain both β-(1 → 3) and β-(1 → 4) linkages. This interpretation was supported by the comparison with three DP4 commercial standards, all containing one β-(1 → 3) linkage (Additional file 1: Figure S1). The presence of one β-(1 → 3) linkage in the oligosaccharide changes the shape of the gas-phase ions and increases the drift time compared to the structure with three consecutive β-(1 → 4) linkages. Similar profiles of ion mobility were observed for the ions having a mass shift of −2 and +16 Da (m/z 687.2 and 705.2, respectively) from the non-modified DP4, which likely correspond to oxidized products (Additional file 1: Figure S2).
The results thus showed that DP4 species released from lichenan and MLG by PaLPMO9H had two main types of structures: one displaying three consecutive β-(1 → 4) linkages, the other encompassing at least one β-(1 → 3) linkage. MLG and lichenan contain a significant proportion of β-(1 → 3) linkages. On average, the ratio of (1 → 3) to (1 → 4) β-linkages is of 1:3 to 1:2 in these two polymers. In barley MLG, cellodextrin units of two (DP3) or three (DP4) adjacent β-(1 → 4) linkages are predominant, while longer cellodextrin sequences contributed to less than 10% of the total polysaccharide chain in the starchy endosperm [23]. If one assumes that DP4 structures of different shapes have comparable ionization efficiency in MS, the peak area in Fig. 2 gives an estimate of the proportion of the two populations of DP4 differentiated by their ion mobility. The DP4 made of three consecutive β-(1 → 4) bonds was released in high proportion following action of PaLPMO9H on MLG and lichenan substrates. DP5 species were also released from MLG and lichenan (Additional file 1: Figure S3). The most abundant peak (dt = 92 bins) falls at the same drift time as the one measured from cellulose. Without excluding that those species arise in part from long cellodextrin stretches in the polymer, their abundance suggests that β-(1 → 3) bonds linking consecutive cellotetraosyl units were cleaved by PaLPMO9H. Thus, in addition to β-(1 → 4) linkages, PaLPMO9H appears able to catalyze the oxidative cleavage of β-(1 → 3) linkages in β-(1 → 4; 1 → 3)-linked glucosidic substrates (MLG and lichenan).
The two DP4 oxidized degradation products (−2 and +16 Da compared to the non-modified form) from lichenan and MLG were further fragmented to confirm their structures. In each case, ion mobility was used to distinguish between the isomers arising from β-(1 → 4) and/or β-(1 → 3) linkages. In accordance with the above discussion, the isoform displaying the lower drift time for each of these ions was attributed to a β-(1 → 4)-linked oligosaccharide while the second isoform (higher drift time) presumably contained a mixture of β-(1 → 3) and β-(1 → 4) bonds. The corresponding tandem MS spectra are displayed in Fig. 3. Fragmentation of the ion at m/z 687.17 (−2 Da compared to the non-modified DP4, Fig. 3c) led to several fragments that are characteristic of a ketone form at the non-reducing end (2,5X1, 1,5X2, 2,5X3). In contrast, the species at m/z 705.18 (+16 Da compared to the non-modified DP4, Fig. 3d) exhibited a double loss of water from the parent ion (indicated as −2 H2O on the fragmentation spectrum) under fragmentation, which is a signature of an oxidized non-reducing end in the gem-diol form, as reported by Isaksen et al. [14]. The fragment 1,5X2, observed at m/z 393.08, further confirmed this structure. Since lichenan are linked through β-(1 → 4) and/or β-(1 → 3) bonds and taking into account the observations we have made previously, the oxidative cleavage by PaLPMO9H may have occurred on position C4 or C3. However, the fragmentation spectra for the two oxidized species (−2 and +16 Da) did not allow the determination of which position was oxidized among the C4 and C3 of the non-reducing end glycoside residue. Yet, for a simplified representation, the oxidation was positioned at C4 in the structures depicted in Fig. 3a, b. The second isoform separated by IM with a higher drift time was also fragmented for the two oxidized species. Similar to the first isoform, fragments indicating a ketone and a gem-diol on the non-reducing end were observed for the parent ions at m/z 687.17 and m/z 705.18, respectively (data not shown). Similar to the reaction products from lichenan, the two isoforms of the species at m/z 687.19 produced from the MLG substrate were found to arise from a ketone at the non-reducing end, while both isoforms of the species at m/z 705.18 were found to correspond to a gem-diol at the non-reducing end. In summary, PaLPMO9H oxidatively cleaved lichenan and MLG substrates, producing both ketone and gem-diols species at the non-reducing end glycoside residue. The exact positioning of the oxidation (C4 or C3) remained undetermined.
PaLPMO9H is active toward β-(1 → 4)-linked glucosidic substrates with branched sidechains
To examine the mode of action of PaLPMO9H on xyloglucan, the tetradecasaccharide XXXGXXXG was used as a model substrate. Named according to the standard linear nomenclature, in which “G” represents an unbranched β-(1 → 4)-linked glucosyl residue and “X” represents β-(1 → 4)-linked Glc bearing an α-(1 → 6)-linked xylosyl branch [24], XXXGXXXG presents a highly decorated backbone consisting of eight glucosyl residues. The MS spectrum of the products of cleavage of the XXXGXXXG preparation by PaLPMO9H (Fig. 1e) showed the presence of minor peaks, whose masses indicated the presence of sidechain galactosylation. They likely arose from a DP15 XyG, which was present as minor impurity (roughly 5%) of the XXXGXXXG preparation as confirmed by both MS and HPAEC-PAD chromatogram of the substrate before incubation with PaLPMO9H (Additional file 1: Figure S4). In the latter structure (likely XXLGXXXG and/or XXXGXXLG), one of the laterally branched xylose bears a β-(1 → 2)-linked galactosyl (represented as “L” [24]).
Tandem MS analyses were performed on the species of m/z 645.16, which uniquely matches a hydrated oxidized XX species (+16 Da from the non-modified oligosaccharide, Fig. 1e). The fragmentation spectrum suggested the coexistence of at least two oxidized species (Fig. 4). First, the double loss of water (indicated as −2.H2O on the fragmentation spectrum) from the precursor ion was found, which indicated a gem-diol at the non-reducing end. The 0,1A2 fragment was also observed. This fragment ion, corresponding to a loss of 46 Da from the parent, was reported by Isaksen et al. [14] as characteristic of an acidic function at the C1 of the reducing end.
These results clearly indicated that PaLPMO9H was able to oxidatively cleave β-(1 → 4)-linked glucose chains even in the presence of extensive lateral branching.
Activity of PaLPMO9H on β-(1 → 4)-linked hetero-polysaccharides
Glucomannans are mainly straight-chain polymers of β-(1 → 4)-linked d-mannose and d-glucose. The product released at 705.18 m/z corresponding to a +16 Da mass shift from the non-modified DP4 was fragmented in tandem MS. The observed fragment ions showed the coexistence of two oxidized species with (i) the loss of 46 Da from the parent ion indicating a C1 oxidation (aldonic acid) at the reducing end, and (ii) a double loss of water from the precursor ion indicating a gem-diol at the non-reducing end (Additional file 1: Figure S5). On the other hand, fragmentation of the −2 Da species showed unambiguously a single species, corresponding to a ketone form at the non-reducing end. It can thus be concluded that PaLPMO9H oxidatively cleaved GM by forming oxidized species both at the reducing end and the non-reducing end. Since the main chain of GM consists of β-(1 → 4)-linked residues, it can be presumed that the oxidized positions are at C1 and C4, respectively.