Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity

Abstract

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

Keywords: auxiliary activity family 10 (AA10); carbohydrate-active enzyme; cellulose; chitin; glycosidic bond cleavage; high-throughput screening; lytic polysaccharide monooxygenase (LPMO); protein stability; renewable energy; substrate specificity.

Figure 1.

Structural overview of targeted residues in ScLPMO10C. A displays a side view of the crystal structure of the catalytic domain of WT ScLPMO10C (PDB code 4OY7 (34)) with the substrate-binding surface facing downwards. The side chains of residues targeted for mutation are shown with green-colored carbons, and carbons in the side chains of the two histidines that coordinate the copper ion (orange sphere) are colored gray. B shows the substrate-binding surface with residue numbering according to the PDB structure, which is used throughout this study. Note that His-35 is the N-terminal residue of the mature protein and that the mutational work was carried out on the full-length enzyme that includes a family 2 CBM connected by a flexible Pro/Thr-rich linker (13). C shows amino acid frequencies encountered at each mutated position (1–5) in multiple sequence alignments of sequences belonging to various LPMO10 subgroups: ScLPMO10C-like (26 sequences, C1-oxidizing, cellulose); MaLPMO10B-like (28 sequences, C1/C4 oxidation of cellulose and C1 oxidation of chitin); and SmLPMO10A-like (49 sequences, C1-oxidizing, chitin). Position 4 (ScLPMO10C_Y111) is exclusively found in some ScLPMO10C-like sequences and is always present with the upstream motif Asn–Trp–Phe in which the first and the last make up positions 2 and 3 in the library. Mutations included in the library are shown in the top right corner of panel C. Panels A and B were made using PyMOL and the graphs in panel C were generated using WebLogo (36). A blue star in A–C marks the location of the second histidine (His-144) of the histidine brace. See Table S1 for the sequences used in the analyses and Fig. S1 for a structural comparison of natural LPMO10s with experimentally-verified different substrate specificities.

Figure 2.

Screening for chitinolytic activity. Chitinolytic activity was assessed using 2 g/liter β-chitin and 1 mm ascorbic acid (reductant) at pH 6.0 and 40 °C. The figure shows RapidFire ESI triple quadrupole MRM chromatograms for M2 (four independent wells, light green) and WT ScLPMO10C (four independent wells, gray). The ESI MRM intensity is given on the y axis, while time (seconds) is on the x axis; note the different scales on the y axis. Chromatograms A–E show the following: A, DP2^ox: ESI MRM m/z 439.2 → m/z 116.0; B, DP3^ox: m/z 643.3 → m/z 625.3; C, DP4^ox: 845.3 m/z → m/z 668.2; D, DP5^ox: m/z 1048.2 → m/z 871.2; E, DP6^ox: m/z 1251.5 → m/z 871.3. Underlying spectral data are shown in Fig. S2. A clone was defined as positive if, for at least one oxidized product, the difference between the average of the green peaks was higher than the average of the gray peaks by at least 10 times the standard deviation in the average for the gray peaks. The picture shows that the developed method failed for DP3^ox, which shows similar signals for M2 and ScLPMO10C.

Figure 3.

Oxidized products generated from chitin. A shows the results of MALDI-ToF MS analysis after a 24-h incubation of the positive control LPMO (SmLPMO10A) with 10 g/liter β-chitin at 40 °C in sodium phosphate (pH 6.0) in the presence of 1 mm ascorbic acid (reducing agent) and displays the C1-oxidized products (aldonic acids and lactones) that can be expected for a chitin-active LPMO. Peaks representing oxidized chito-oligomers are marked with their m/z value (see A for details). B shows a zoom-in view of the hexamer cluster displaying SmLPMO10A-generated products within the 1250 to 1320 m/z range. The peaks that are marked with an asterisk represent potassium adducts, which are absent in the spectra for the ScLPMO10C mutants due to different storage buffers (SmLPMO10A was stored in potassium phosphate (pH 6.0), and the ScLPMO10C mutants in sodium phosphate (pH 6.0)). C shows a zoom-in view of the hexamer cluster area for WT ScLPMO10C and shows no oxidized products. D–F display zoom-in views of the hexamer cluster area for mutants M2, M5, and M18, respectively. Arrows in B, D–F indicate minor signals at m/z = 1256 and m/z = 1260 that are more prominent in the mutants (relative to the signals for regular oxidized products) compared with SmLPMO10A.

Figure 4.

Time course of chitin degradation. SmLPMO10A, ScLPMO10C WT, and the three chitin-active ScLPMO10C variants M2, M5, and M18 were incubated at 1 μm with 10 g/liter β-chitin at 40 °C and pH 6.0 in the presence of 1 mm ascorbic acid (reducing agent) for 4 h. A chitinase (SmChiC) was also added at 0.25 μm to obtain more rapid solubilization of LPMO-generated chain ends from the insoluble substrate. The reaction was stopped by vacuum filtering, and the soluble oxidized products were converted to oxidized dimers by treatment with chitobiase prior to analysis. All reactions were performed in triplicate. The error bars show ± S.D. (n = 3).

Figure 5.

Mutational effects on thermostability. The plots show the apparent T_m of copper-saturated SmLPMO10A, ScLPMO10C WT, M2, M5, and M18. The derivative of the fluorescence signal (−dRFU/dT, where RFU is relative fluorescence units) is plotted as a function of the temperature (39). The reactions contained 0.1 g/liter LPMO and were heated from 25 to 99 °C, at a rate of 1.5 °C/min, in the presence of a fluorescent dye (SYPRO Orange). The scans were performed four times for each protein, and the figures show a typical scan. All apparent T_m values had standard deviations below ±0.1 °C.

Figure 6.

H₂O₂ as co-substrate. A shows the amount of solubilized oxidized sites produced by SmLPMO10A (black line), M18 (dashed black line), and ScLPMO10C WT (dotted gray line) over 60 min in a reaction with 10 g/liter β-chitin in 50 mm sodium phosphate (pH 6.0), which were supplied with 100 μm AscA and 15 μm H₂O₂ every 15 min (indicated by arrows). The enzyme concentration was 1 μm in all cases. Aliquots were withdrawn between H₂O₂ additions (at 7.5, 22.5, 37.5, and 52.5 min) and immediately prior to addition of fresh H₂O₂ (at 0, 15, 30, and 45 min). The apparent plateaus in product levels between the intermediate sampling points (e.g. at 7.5 min) and the subsequent point of addition of fresh H₂O₂ and AscA (e.g. at 15 min) are because all the H₂O₂ added at the preceding addition (in this example, 0 min) was consumed at the intermediate sampling point (7.5 min). The lines connecting the points are drawn for illustration purposes only, and actual reaction rates were higher than suggested by the slopes of the ascending lines. B shows control reactions related to A. In the reactions labeled “without H₂O₂,” water was added to the reactions instead of H₂O₂. The graph also shows H₂O₂-driven product formation by WT ScLPMO10C (same curve as in A). Note the difference in the scale of the y axis between A and B; the product levels shown in B are very low, making quantification difficult. C shows the total amounts of oxidized sites at the end of the reactions shown in A (i.e. after 60 min). D shows the effect of varying the amount of added ascorbic acid in the reactions displayed in A (addition of 15 μm H₂O₂ + varying amounts of AscA every 15 min) for M18 and shows that essentially similar product yields were obtained when using a 10 times lower AscA concentrations compared with the reactions shown in A. The control reaction to the left shows that the LPMO is essentially inactive in the absence of added reductant. All reactions were performed in triplicates, and the error bars show ± S.D. (n = 3). Prior to analysis, soluble oxidized products were converted to oxidized dimers by treatment with a chitobiase. The total amount of oxidized products was determined by treating the complete reaction mixture with a mixture of chitinases and chitobiase, which leads to complete solubilization of the chitin and to conversion of all oxidized products, soluble or insoluble (i.e. still part of the chitin), to chitobionic acid.

Figure 7.

Effect of single mutations on chitin degradation by M18. ScLPMO10C WT, M18, and the four M18 mutants were incubated at 1 μm with 10 g/liter β-chitin at 40 °C in 50 mm sodium phosphate buffer (pH 6.0) in the presence of 1 mm ascorbic acid for 24 h. SmChiC was also added, at 0.25 μm, to obtain more rapid solubilization of LPMO-generated oxidized chain ends from the insoluble substrate. The reaction was stopped by filtration, and soluble oxidized products were degraded to oxidized dimers with chitobiase prior to analysis. All reactions were performed in triplicates. The error bars show ± S.D. (n = 3).

Figure 8.

Residual cellulolytic activity. ScLPMO10C WT and mutants were incubated at 1 μm with 5 g/liter PASC and 1 mm ascorbic acid at 40 °C and in 50 mm sodium phosphate (pH 6.0). A shows oxidized products after ∼24 h of incubation, varying from the dimer (GlcGlc1A) to the octamer (Glc₇Glc1A), generated by ScLPMO10C WT, M2, M5, and M18. The background signal of the substrate (PASC) incubated with LPMO in the absence of reductant is also shown. Note that the size of the peaks of the WT chromatogram was reduced by 25% before making the plot. Regions where C4-oxidized products would elute are marked. B shows the amount of soluble oxidized sites in aliquots that were withdrawn after 1 and 4 h of incubation; in this case oxidized products were quantified after degrading them to a mixture of oxidized dimers (GlcGlc1A) and trimers (Glc₂Glc1A) only, by treatment with the endoglucanase TfCel5A. Relative activities were calculated relative to the amount of product generated by the WT enzyme after 4 h (100%). Testing of the cellulolytic activity of M18 with Avicel gave similar results. All reactions were performed in triplicate, and the error bars show ± S.D. (n = 3).

Figure 9.

Comparison of chitinolytic and cellulolytic activity. Comparison of solubilized oxidized products generated in 4 h by 1 μm M18 or 1 μm ScLPMO10C WT from PASC (5 g/liter) or β-chitin (10 g/liter). 100% activity for ScLPMO10C WT on PASC is equal to 218.6 μm (sum of GlcGlc1A and Glc₂Glc1A, obtained after cellulase treatment of soluble products as described under “Experimental procedures”). 100% activity for M18 on β-chitin is equal to 20.6 μm (GlcNAcGlcNAc1A, obtained after chitobiase treatment of soluble products as described under “Experimental procedures”). The solubilized oxidized products were converted to oxidized dimers before quantification. Note that quantification of products generated from β-chitin by the WT enzyme is inaccurate as the product levels are close to the lower detection limit. All reactions were performed in triplicates, and the error bars show ± S.D. (n = 3).

Figure 10.

Models of substrate binding by LPMOs. A shows a model (extracted from MD simulations performed on β-chitin (30)) of SmLPMO10A interacting with GlcNAc₇. The side chains of the copper-binding histidines and four of the five residues that were targeted for mutagenesis in cellulose-active ScLPMO10C are shown as sticks (the shown residues correspond to Tyr-79, Asn-80, Phe-82, and Trp-141 in ScLPMO10C; Tyr-111 in ScLPMO10C does not have an analogue in SmLPMO10A). B shows a model of M18 interacting with GlcNAc₇ that was generated by superposing the crystal structure of the catalytic domain of ScLPMO10C onto the existing SmLPMO10A–chitin model. The mutations found in M18 were introduced using the mutagenesis tool in PyMOL, and rotamers with the highest probabilities were selected. C shows the same model for WT ScLPMO10C, showing that two aromatic residues (Tyr-111 and Trp-141), which are both mutated in M18, may restrict contact with the chitin substrate. B–D highlight two loop regions, Ser-109–Pro-110–Tyr-111 and Ser-182–Pro-183–Gly-184–Thr-185 (red in B–D) that are extended in ScLPMO10C, relative to natural chitin-active LPMOs, and that may interfere with chitin binding. D shows a superposition of WT ScLPMO10C (green and red) and SmLPMO10A (gray) bound to β-chitin (yellow). The structures shown in D are rotated 90° clockwise relative to the structures shown in A–C.