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An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides

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Science  08 Oct 2010:
Vol. 330, Issue 6001, pp. 219-222
DOI: 10.1126/science.1192231

Abstract

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H218O or 18O2. There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass.

The transition to a more environment-friendly economy has spurred research on enzymes capable of efficiently degrading recalcitrant polysaccharides, such as cellulose and chitin (Fig. 1A), for the production of biofuels (1). Traditionally, enzyme systems capable of degrading such polysaccharides are considered to consist of endo-acting enzymes that cut randomly in the polysaccharide chain and processive exo-acting enzymes (chito- or cellobiohydrolases), which degrade the polymers from chain ends. All these enzymes are hydrolytic and are referred to as glycoside hydrolases. Although this model is generally accepted, it remains difficult to understand how the glycoside hydrolases could act on a polysaccharide chain in its crystalline environment, and biochemists have speculated about the existence of a substrate-disrupting factor that could make the crystalline substrate more accessible to hydrolytic enzymes (2).

Fig. 1

(A) Repetitive disaccharide unit in cellulose and chitin. (B) Crystal structure of CBP21 (10) (PDB ID: 2BEM). The side chains of conserved histidine residues on the flat binding surface are shown in orange-colored stick representation. The binding surface has been identified previously using site-directed mutagenesis and binding studies (10) and is indicated by a black line. (C) Crystal structure of GH61E from Thielavia terrestris (7) (PDB ID: 3EII). The side chains of conserved histidine residues are shown in cyan-colored stick representation. (D) Detailed view of the conserved arrangement of the two histidines and the N-terminal amino group in CBP21 (light brown) and GH61E (white), superimposed using PyMol. (B), (C), and (D) were made using PyMol.

Recently, it was discovered that microorganisms that break down chitin, a crystalline analog of cellulose occurring in the shells of insects and crustaceans, indeed produce a protein that increases substrate accessibility and potentiates hydrolytic enzymes (3) (Fig. 1B). These proteins are classified as carbohydrate-binding modules (CBMs) and belong to family CBM33 as defined in the Carbohydrate Active Enzymes (CAZy) database (4, 5). The first example of such a protein is CBP21 (CBP for chitin-binding protein), produced by the chitinolytic bacterium Serratia marcescens. As another example, two CBM33-containing proteins from Thermobifida fusca potentiate chitin hydrolysis by chitinases and cellulose hydrolysis by cellulases (6). Genes putatively encoding CBP21-like proteins are abundant in bacteria and viruses but are rare in eukaryotes. Fungi produce proteins classified as family 61 glycoside hydrolases (GH61) that act synergistically with cellulases (7) and are structurally similar to CBM33 proteins (7, 8) (Fig. 1, B and C). The structural similarity includes a diagnostic conserved arrangement of the N-terminal amino group and two histidines that bind a metal ion (Fig. 1D). One of these histidines (His28/His19 in Fig. 1D) is the N-terminal residue of the mature protein that results after proteolytic processing of the signal peptide during secretion. So far, the mechanisms employed by CBP21-like and GH61-like proteins have remained elusive.

We show here that CBP21 is an enzyme that catalyzes cleavage of glycosidic bonds in crystalline chitin, thus opening up the inaccessible polysaccharide material for hydrolysis by normal glycoside hydrolases. This enzymatic activity was first discovered when we detected traces of previously unidentified chitooligosaccharides upon incubation of β-chitin nanowhiskers with CBP21 (Fig. 2A) (9). The products were identified as chitin oligosaccharides with a normal sugar at the nonreducing end and an oxidized sugar, 2-(acetylamino)-2-deoxy-d-gluconic acid (GlcNAcA), at the other end (Fig. 2B and fig. S1). Addition of reductants dramatically increased the efficiency of the reaction (Fig. 2, C and D), which enabled the breakdown of large crystalline β-chitin particles by CBP21 alone (Fig. 2), with the release of a range of oxidized products (figs. S2 and S3 and below). In the presence of a reductant such as ascorbic acid, CBP21 boosted chitinase efficiency to much higher levels than previously observed (Fig. 2D) (3). Biotechnological applications could take advantage of the ability to increase CBP21 activity by adjusting the reaction conditions.

Fig. 2

(A) Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) spectrum showing oxidized soluble chitooligosaccharides generated by CBP21 acting on β-chitin whiskers. (B) Oxidized chitooligosaccharide with a GlcNAcA moiety. (C) Change in the polymeric material on treatment with CBP21 in the presence of ascorbic acid: 2.0 mg/ml β-chitin was treated with 1.0 μM CBP21 in 20 mM Tris-HCl, pH 8.0, in the presence (left vial) or absence (right vial) of 1.0 mM ascorbic acid for 24 hours at 37°C. After the incubation, which leads to the oxidation of ~7.6% of the sugars (see main text), the glass vials were shaken vigorously. After a 1-min rest for particle sedimentation, the picture was taken. (D) Efficiency of β-chitin (0.45 mg/ml) degradation by 0.5 μM ChiC in the presence of 1.0 μM CBP21 and 1.0 mM ascorbic acid at pH 8.0 (magenta line on diamonds). Parallel reactions containing β-chitin and ChiC in the presence (red line on squares) or absence (yellow line on circles) of CBP21 (same concentrations as above) and without ascorbic acid are also shown. Data points for the incubation of β-chitin with ChiC and ascorbic acid overlap with the yellow points; this reaction is shown in fig. S7. Data are means ± SD (n = 3); error bars (not visible for every point) indicate SD. (E) MALDI-TOF MS spectrum of products obtained after incubating β-chitin (2.0 mg/ml) with 1.0 μM AnCDA, 1.0 μM CBP21, and 1.0 mM ascorbic acid for 16 hours, showing oxidized deacetylated chitooligosaccharides (all major products contain two acetylated sugars). A control reaction without CBP21 did not yield soluble products (results not shown). (F) MALDI-TOF MS spectrum of products obtained after incubating β-chitin (2.0 mg/ml) with 1.0 μM AnCDA and 0.5 μM ChiC for 8 hours, showing deacetylated chitooligosaccharides (all major products contain one acetylated sugar). MS peaks are labeled by observed atomic mass (listed in table S1) and the degree of polymerization (DP) of the oligosaccharide. Labels always refer to the peak of highest intensity in the respective cluster, which comprises several adducts; in (A) [M+Na]+ ions are labeled; in (E) and (F), [M+H]+ ions are labeled. “ox” indicates the presence of a GlcNAcA moiety. Asterisks indicate a deacetylated product. In (A), (E), and (F), 100% relative intensity represents 2.5 × 104, 1.1 × 103, and 5.4 × 104 arbitrary units (a.u.), respectively. See fig. S1 for additional experiments to verify products and table S1 for an overview of possible ions and masses.

If CBP21 acts randomly on crystalline surfaces, one would expect generation of longer oligosaccharides, which are difficult to detect owing to their low solubility. The majority of soluble products generated by CBP21 in the presence of a reductant had a degree of polymerization (DP, i.e., the number of sugar moieties) below 10 (Fig. 2A and fig. S2). We therefore exploited a newly cloned chitin deacetylase from Aspergillus nidulans (AnCDA) (9) to increase the solubility of longer chitin fragments by deacetylation. This approach revealed the formation of chitin fragments with high DP, in the presence of CBP21 (Fig. 2E) or an endochitinase (ChiC) (Fig. 2F). Both CBP21 and ChiC generated long products, indicative of an “endo”-type of activity. Two important features stand out. First, all CBP21 products are oxidized, which confirms the observation that the cleavage of glycosidic bonds by CBP21 includes an oxidative step. Second, whereas the products released by ChiC represent a continuum of lengths (Fig. 2F), the products released by CBP21 are dominated by even-numbered oligosaccharides (Fig. 2E and figs. S2 to S4). Thus, ChiC tends to cleave any glycosidic bond, whereas CBP21 shows a strong preference for cleaving every second glycosidic bond. Keeping in mind the disaccharide periodicity in the substrate (Fig. 1A), this observation implies that ChiC approaches single polymer chains from “any side,” whereas CBP21 must approach the substrate from one side. The latter is consistent with polysaccharide cleavage in the context of an intact crystalline structure.

The CBP21-mediated cleavage mechanism was probed in more detail by isotope-labeling. Experiments in H218O showed that one of the oxygen atoms introduced at the oxidized chain end comes from water (Fig. 3A). The only plausible source for the second oxygen was molecular oxygen, and this was confirmed by experiments performed in 18O2 saturating conditions (Fig. 3, B and C). Removal of dissolved molecular oxygen in the reaction solution inhibited CBP21 activity (fig. S5), which confirmed the requirement for molecular oxygen for catalysis. Thus, the reaction catalyzed by CBP21 comprises a hydrolytic step and an oxidation step, as summarized in Fig. 3D. We suggest naming CBP21 a “chitin oxidohydrolase.”

Fig. 3

MALDI-TOF MS analysis of products detected after treating 2.0 mg/ml β-chitin with 1.0 μM CBP21 and 1.0 mM ascorbic acid in Tris-buffered H218O pH 8.0 (A) or in Tris-buffered H216O, pH 8.0, saturated with 18O2 (B). Labels in (A) and (B) refer to [M+Na]+ ions (see table S1). All major products show a mass increase of 2 atomic mass units (amu) compared with reactions performed in solutions not containing isotope-labeled water or molecular oxygen (see Fig. 2A and fig. S2). (C) Adducts of the oxidized hexameric product shown in (B). Note the small amount of non–isotope-labeled product (indicated by the arrow), most likely resulting from the initial stage of the reaction where 16O2 was still present (before 18O2 saturation) (9). In (A), (B), and (C), 100% relative intensity represents 6.8 × 103 and 2.0 × 103 a.u. respectively. (D) Scheme for the enzymatic reaction catalyzed by CBP21. In the final oxidized product, one oxygen comes from molecular oxygen (blue) and one from water (red).

CBP21 catalysis was found to be inhibited by EDTA, and activity could be restored by adding divalent cations such as Mg2+ or Zn2+ (fig. S6), which may bind to the conserved histidine motif (Fig. 1D). Note that the activity of GH61 proteins also depends on similar divalent cations (7). Structural studies of both CBP21 (10) and GH61 proteins (7, 8) show considerable structural plasticity in the metal-binding site, explaining why the metal-binding site is promiscuous and why the need for divalent cations is rather unspecific [as shown in fig. S6 and (7)]. Mutation of the second histidine (His114 in CBP21 and His86 in GH61E from Thielavia terrestris) in the metal-binding motif knocked out activity of both proteins [fig. S7 and (7), respectively]. As CBP21 is a redox enzyme, it is remarkable that the metal ions activating CBP21 are not redox active. Perhaps the metal ion’s primary role lies in stabilization of the active-site structure and/or positioning of a hydrolytic water molecule.

Using newly developed methods for quantification of oxidized products that are described in detail in the supporting online material, we were able to estimate the speed and degree of oxidation under various conditions (fig. S8). When CBP21 acts alone on β-chitin under optimal conditions, the oxidation rate is on the order of 1/min, and the maximum extent of oxidation is about 7.6% of the sugars. Simultaneous degradation of β-chitin with CBP21 and ChiC under optimal conditions led to complete substrate solubilization and oxidation of ~4.9% of the sugars. It must be noted that, in nature, enzymes such as CBP21 normally act simultaneously with at least one, and in the case of S. marcescens, three chitinases (11).

Control experiments confirmed the conclusion that formation of oxidized products only occurs in the presence of CBP21 and crystalline substrates. The presence of reductants alone did not yield oxidized products (fig. S9) and did not potentiate or inhibit chitinase action (Fig. 2D and fig. S7). When incubated under optimal conditions with hexameric N-acetylglucosamine, neither degradation products nor oxidized oligosaccharides were observed (fig. S10). We also considered the possibility that CBP21 might work without directly interacting with the cleaved bond, because one could envisage a CBP21-induced “destructuring” mechanism making the substrate more accessible for the action of reactive oxygen species generated in a CBP21-independent manner, e.g., by Fenton chemistry. However, we could not detect any soluble products after subjecting β-chitin to Fenton chemistry (fig. S11). All these experiments are consistent with CBP21’s actively participating in the cleavage reaction and the oxidative step.

CBP21 activity is strongly inhibited by cyanide, a known O2 mimic, but not by azide, a known inhibitor of heme proteins (fig. S5). Superoxide dismutase did not inhibit CBP21 activity, whereas catalase had only a minor inhibitory effect. Several reductants capable of functioning as electron donors boosted the activity of CBP21 (Fig. 2D and fig. S3). The experimental data indicate that the oxidation step catalyzed by CBP21 is cofactor-independent and depends on an external electron donor. The insensitivity for superoxide dismutase indicates that oxygen is activated on the enzyme, where it would be shielded from the solvent. The strong inhibition by cyanide supports the crucial role of the oxidative step. Cofactor-independent oxygenases have been described before, but these enzymes are normally thought to use conjugated carbanions in the substrates as electron donors (12), a mechanism that is not likely in the case of a polysaccharide substrate. If the oxidation step was to happen first, this would imply that CBP21 catalyzes cofactor-independent oxygenation of a saturated carbon, which is unprecedented and perhaps not very likely. On the other hand, such a mechanism could yield an intermediate product (for example, an ester bond) that may be more prone to hydrolysis than the original glycosidic bond. Alternatively, the hydrolytic step could occur first, which would imply that CBP21 is capable of hydrolyzing glycosidic bonds in a crystalline environment using a hitherto unknown mechanism. Such a hydrolytic step would require some degree of substrate distortion (13, 14), which seems challenging in a crystalline packing. However, in favor of this mechanism, the subsequent oxidation of the resulting sugar aldehyde (“reducing end”) is more straightforward than oxidation of a saturated carbon. Clearly, further experiments are needed to unravel mechanistic details of the remarkable reaction catalyzed by CBP21.

CBP21 introduces chain breaks in what probably are the most inaccessible and rigid parts of crystalline polysaccharides, and its mode of action differs fundamentally from the mode of action of glycoside hydrolases. Glycoside hydrolases are designed to host a single “soluble” polysaccharide chain in their catalytic clefts, and their affinity and proximity to the crystalline substrate tend to be mediated by nonhydrolytic binding domains. In contrast, CBP21 binds to the flat, solid, well-ordered surface of crystalline material and catalyzes chain breaks by a mechanism that results in oxidation of one of the new chain ends. The chain break will result in disruption of crystalline packing and increased substrate accessibility, an effect that may be enhanced by the oxidation of the new chain end that disrupts the normal chair conformation of the sugar ring and introduces a charge.

The enzyme activity demonstrated in this study is difficult to identify because products have low solubility and potentially a high tendency to remain attached to the crystalline material. Based on the structural homology and other similarities discussed above, we propose that GH61 proteins may have the same activity as CBP21, but the even lower product solubilities and higher crystalline packing of cellulose compared with chitin (15) make direct detection of this activity very challenging. However, a first glimpse of the potential of GH61 proteins for cellulose conversion has been presented recently (7). The dependency of these enzymes on the presence of molecular oxygen and reductants provides guidelines for process design.

Supporting Online Material

www.sciencemag.org/cgi/content/full/330/6001/219/DC1

Materials and Methods

Figs. S1 to S12

Table S1

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank A. C. Bunæs for technical assistance and G. Vriend for helpful discussions. This work was supported by Norwegian Research Council grants 171991/V40, 164653, 186946/I30, and 190877/S60.
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