Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (PoGH74cat) revealed a highly specific, processive endo-xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform — the first in GH74 extending from subsites −4 to +6 — and unique mobile active-site loops. Site-directed mutagenesis revealed that glycine-476 was uniquely responsible for the promiscuous backbone-cleaving activity of PoGH74cat; replacement with tyrosine, which is conserved in many GH74 members, resulted in exclusive hydrolysis at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity.

Introduction

The replacement of fossil petroleum by sustainable resources remains one of the greatest challenges of the 21st century. The utilization of plant biomass can significantly contribute to this transition, providing an abundant and renewable source of carbohydrates that can be converted through chemical and biological processes into biofuels or a wide range of bioproducts. Compared with dedicated crops, lignocellulosic biomass is particularly advantageous because it does not compete with the food sector. However, a major technological obstacle is the recalcitrance of the raw material due to its complex structure [1]. The plant cell wall is composed mainly of cellulose organized in microfibrils that are embedded in a matrix of various proteins, hemicellulosic and pectic heteropolysaccharides and polyphenolic lignins [2]. This variable structure and composition creates a barrier for the saccharification of the plant cell wall but also constitutes a supply for the production of a wide range of co-products, notably derived from the hemicellulosic fraction.

The xyloglucans (XyGs) comprise a prominent family of hemicelluloses that are ubiquitous in the primary wall of land plants, constituting up to 20% of the dry weight of the cell wall (e.g. the primary cell wall of dicotyledons) [3,4]. XyGs are thought to coat and tether cellulose microfibrils within a composite matrix [5]. Enzymatic XyG hydrolysis increases cellulose digestibility [6,7], which underscores their structural importance in the plant cell wall. XyGs are also found as storage polysaccharides in some seeds, e.g. tamarind [8], and therefore represent important agricultural byproducts that have applications in the food, biomaterial and medical sectors [911].

Structurally, XyGs are composed of a β-(1,4)-linked glucopyranosyl backbone that is highly substituted with side chains of varying length and pattern depending on plant and tissue origin [12]. In dicots, typically three out of four glucose units of the backbone are decorated by xylopyranosyl units, forming XXXG type-repeating motifs, where G represents an unbranched backbone glucosyl unit and X represents a branched [α-Xyl-(1,6)]-β-Glc-(1,4) unit (nomenclature according to ref. [13]). The xylopyranosyl branch can be further derivatized by a β-(1,2)-linked galactopyranosyl residue; this trisaccharide unit is denoted L. XyGs can be substituted with a variety of other side chains, e.g. arabinofuranosyl units and fucopyranosyl units [12].

Endo-xyloglucanases, which catalyze the cleavage of the XyGs backbone (EC 3.2.1.151), are currently found in glycoside hydrolase (GH) families GH5, GH9, GH12, GH16, GH44, and GH74 in the Carbohydrate-Active enZymes (CAZy) classification [1416]. Of these, family GH74 is distinguished by fewer sequence members, an essentially singular specificity for XyGs [1735], and a characteristic tertiary structure comprised of two 7-bladed β-propeller domains that form a large interfacial cleft to accommodate the bulky polysaccharide [17,3639]. Structure–activity relationships among characterized GH74 members, including determinants of endo- versus exo- (EC 3.2.1.150) activity, have recently been reviewed [40].

Endo-xyloglucanases from GH74 can hydrolyze the regular structure of ‘XXXG-type’ XyGs at the anomeric center of the unbranched glucosyl (‘G’) unit [17,23,37], although some cleave the backbone at more sterically encumbered positions, e.g. between two ‘X’ units [22,24,27]. In addition to those exhibiting classical endo-dissociative activity [27,34], in which the enzyme attacks the XyG backbone in a stochastic fashion followed by the release of both hydrolysis products, some GH74 members have been shown to act via an endo-processive mode [25,27,30]. Such endo-processive xyloglucanases release short (e.g. Glc4-based) xyloglucan oligosaccharides (XyGOs) by multiple sequential hydrolytic events following a single polysaccharide chain-binding event. Processivity is a hallmark of cellobiohydrolases that possess active site tunnels [41], yet is less intuitive for endo-glycanases with open active-site clefts, such as GH74 members. Seminal work by Yaoi and coworkers has highlighted the importance of specific active-site tryptophan residues to the processivity of a Paenibacillus sp. GH74 endo-xyloglucanase [30]. However, a full molecular understanding of the phenomenon of processivity in GH74 has been limited by a lack of concordant biochemical and experimental enzyme structural data.

Gram-positive bacteria from the genus Paenibacillus (phylum Firmicutes) have found industrial and agricultural applications due to their ability to fix nitrogen in the soil [42], and appear to have a large enzymatic arsenal for cellulose and hemicellulose degradation [43]. Paenibacillus odorifer was isolated from the wheat rhizosphere [44], and its genome encodes for 270 CAZymes, including a single GH74 module contained within a multimodular gene product (NCBI accession number AIQ73809), PoGH74 (nomenclature according to [45]). Here, we performed an in-depth characterization of the catalytic module, PoGH74cat, via a combination of enzyme kinetic and product analysis, site-directed mutagenesis, and protein crystallography. Our results provide new insights into the structural basis of xyloglucan recognition and catalysis by GH74 enzymes, including the contribution of individual active-site amino acids to processivity and backbone cleavage pattern.

Materials and methods

Bioinformatics and phylogenetic analyses

The full-length protein (PoGH74cat-X2-X2-X2-CBM3) encoded by the locus tag PODO_RS11130 in the Paenibacillus odorifer (DSM15391) genome was screened for the presence of a signal peptide using SIGNALP, version 4.0 [46]. The modular architecture was obtained from BLASTP analysis [47]. Screening for similar proteins was performed using BLASTP against non-redundant protein sequences and the CAZy database [15]. Alignments with representative GH, X2, and CBM modules were achieved using Clustal Omega [48] and ESPript software [49].

Cloning and site-directed mutagenesis

Paenibacillus odorifer gDNA (DSM15391) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Germany). cDNA encoding PoGH74cat was PCR amplified from gDNA using the high-fidelity Q5 DNA polymerase (New England Biolabs) and specific primers (PCR primers are listed in Supplementary Table S1). The PCR products were designed such that only the GH74 catalytic module was expressed, removing the signal peptide and other modules (X2 and CBM3). The sequence encoding for the GH74 catalytic domain was flanked by ligation-independent cloning (LIC) adaptors following the recommendations given in ref. [50]. LIC was performed in the vector pMCSG53 as described in ref. [50] to fuse the recombinant proteins with an N-terminal 6× His-tag with a TEV cleavage site connecting the two modules. PoGH74cat(D70A) (catalytic base), PoGH74cat(G476Y) (−1 subsite), PoGH74cat(W406A) (+2 subsite), PoGH74cat(W347A) (+3 subsite), PoGH74cat(W348A) (+5 subsite), PoGH74cat(Y372A) (+6 subsite), PoGH74cat(Δ642–651) and PoGH74cat(Δ671–675) were generated using the PCR-based QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.) in accordance with the manufacturer's instructions and using pMCSG53::PoGH74cat as a template. Primer sequences are provided in Supplementary Table S1.

Gene expression and protein purification

Constructs were individually transformed into chemically competent Escherichia coli BL21 DE3 cells. Colonies were grown on LB solid media supplemented with ampicilin (100 µg/ml). An isolated colony of the transformed E. coli cells was inoculated in 5 ml of LB medium containing ampicilin (100 µg/ml) and grown overnight at 37°C with rotary shaking at 200 r.p.m. The preculture was used to inoculate 500 ml of ZYP5052 autoinducing medium [51] containing ampicilin (100 µg/ml). The culture was grown at 37°C for 4.5 h and transferred to 16°C for overnight culture with rotary shaking at 200 r.p.m until reaching an OD600nm of approximately 11. The culture was then centrifuged at 2220g for 30 min, and the pellet was resuspended in 10 ml of 50 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 20 mM imidazole and frozen at 20°C. Frozen cells were thawed and lysed by the addition of lysozyme (0.5 mg/ml) and benzonase (25 U) and incubation at 37°C for 1 h. In addition, cells were disrupted by sonication and the cell-free extract was separated by centrifugation at 4°C (23000g for 45 min).

Recombinant proteins were purified from the cell-free extract in an Akta Purifier FPLC system using a Ni2+ affinity column. A gradient up to 100% elution buffer (50 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, and 500 mM imidazole) was applied in order to elute proteins from the Ni2+ affinity column. The purity of the recombinant proteins was determined by visualizing fractions by SDS–PAGE. Pure fractions were pooled, concentrated, and buffer-exchanged against 50 mM sodium phosphate buffer, pH 7.0. The final purification step was performed on a size exclusion Superdex 200 column eluted with 50 mM sodium phosphate buffer, pH 7.0. Protein concentration was estimated using an Epoch Micro-Volume Spectrophotometer System (BioTek, Inc., Winooski, VT, U.S.A.) at 280 nm. Protein identities were confirmed by intact MS [52]. BoGH5 was produced and purified following the procedure described in ref. [11].

Carbohydrate sources

Tamarind seed xyloglucan, konjac glucomannan, barley β-glucan, wheat flour arabinoxylan, and beechwood xylan were obtained from Megazyme (Bray, Ireland). Hydroxyethyl-cellulose was purchased from Amresco (Solon, OH, U.S.A.) and carboxymethyl cellulose was from Acros Organics (Morris Plains, NJ, U.S.A.). AZCL-xyloglucan was acquired from Megazyme (Bray, Ireland).

Glc4-based standard xyloglucan oligosaccharides (XyGOs) (XXXG, XLXG, XXLG, and XLLG) were prepared from tamarind seed xyloglucan (Megazyme, Ireland) as described previously [53].

Carbohydrate analytics

High-performance anion-exchange chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) was performed on a Dionex ICS-5000 DC HPLC system with CHROMELEON, version 7 (Dionex Corp., Sunnyvale, CA, U.S.A.) using a Dionex Carbopac PA200 column. Solvent A was distilled water, solvent B was 1 M sodium hydroxide, and solvent C was 1 M sodium acetate. The gradient used was: 0–5 min, 10% solvent B and 3.5% solvent C; 5–12 min, 10% solvent B and a linear gradient from 3.5–30% solvent C; 12–12.1 min, 50% solvent B and 50% solvent C; 12.1–13 min, an exponential gradient of solvent B and solvent C back to initial conditions; and 13–17 min, initial conditions.

Matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Bruker Daltonics Autoflex System (Billerica, MA, U.S.A.). The matrix, 2,5-dihydroxy benzoic acid (DHB), was dissolved in 50% methanol in water to a concentration of 20 mg/ml. Oligosaccharide samples were mixed 1 : 1 (v/v) with the matrix solution. Five microliters of this solution were placed on a Bruker MTP 384 ground steel MALDI plate and left to air dry for 30 min prior to analysis.

Enzyme kinetics and product analysis

Enzymatic assays on AZCL-xyloglucan were performed in triplicate in 1 ml of 50 mM sodium phosphate buffer, pH 7.0, using 1 mg/ml of the substrate and 150 U of PoGH74cat (2.5 µg/ml) and BoGH5 (8 µg/ml) [11], or no enzyme for the negative control. At regular intervals (10, 30, 60, and 120 min), the reaction was centrifuged (10 000g for 2 min) and 180 µl of the supernatant were transferred into a 96-well flat bottom microplate for A590nm measurement.

For all the other enzyme assays on polysaccharides, the activity was determined using the BCA assay as described recently [54]. Substrate specificity was determined in 50 mM sodium phosphate buffer, pH 7.0, using 0.5 mg/ml of the substrate and 1 µg/ml of enzyme overnight at 37°C. The optimum temperature was determined in 50 mM sodium phosphate buffer, pH 7.0, using tamarind seed XyG at a concentration of 0.5 mg/ml and PoGH74cat at a concentration of 0.2 µg/ml at temperatures ranging from 25°C to 65°C. The optimum pH was established at 37°C in 50 mM citrate buffer, pH 3.0, 4.0, 5.0, 5.5, and 6.0, or 50 mM sodium phosphate buffer, pH 6.0, 6.5, 7.0, and 8.0. To test PoGH74cat thermostability, the enzyme was preincubated at different temperatures (37, 41, 46, 51, and 55°C) and the activity of PoGH74cat was measured in 50 mM sodium phosphate buffer, pH 6.0, after 0 min, 15 min, 30 min, 1 h, 2.5 h, 4 h, and 24 h of incubation using tamarind seed XyG at a concentration of 0.5 mg/ml and PoGH74cat at a concentration of 0.2 µg/ml.

To determine Michaelis–Menten parameters of PoGH74cat and its variants for XyG and HE-cellulose, different concentrations of substrate solutions were used over the range of 0.02–2 mg/ml for XyG and 0.05–8 mg/ml for HE-cellulose. The reactions were performed at 37°C in 50 mM sodium phosphate buffer, pH 6.0, using 0.1 µg/ml of the enzyme.

For viscometric assays, 200 µl of diluted enzymes [final concentration of 60 ng/ml for BoGH5, PoGH74cat, PoGH74cat(W347A), PoGH74cat(Y372A) and PoGH74cat(W406A) and 10 ng/ml for PoGH74cat(W348A)] were added to 19.8 ml 0.8% tamarind seed XyG in 50 mM sodium phosphate buffer, pH 6.0, at 30°C. The flow time of reaction mixtures was determined using an Ubbelohde viscometer (Size 1, K = 0.00965, Q Glass Company, Inc., U.S.A.). Flow times were normalized to that of 50 mM sodium phosphate buffer, pH 6.0, according to the equation (tsample − tbuffer)/tbuffer. Reducing sugar concentration was determined by the BCA assay. The degree of hydrolysis at each time point was calculated by comparison with complete digestion of the substrate with excess enzyme and incubation time (designated as 100% hydrolysis).

To determine the degradation products released by PoGH74cat and its variants, tamarind seed XyG was incubated at 37°C in 50 mM sodium phosphate buffer, pH 6.0, at a concentration of 0.5 mg/ml in the presence of enzyme (1 µg/ml for all the others). After various incubation times (0, 5, 10, 30 and 60 min), 100 µl of reaction were sampled and transferred into 20 µl of 1 M NaOH. The reaction solution was then applied to HPAEC-PAD analysis. Limit-digestion products were obtained similarly after 72 h of reaction using 10 µg/ml of enzyme and 0.1 mg/ml of tamarind seed XyG.

X-ray crystallography

For the PoGH74cat apoenzyme and XLX complex, the selenomethionine-substituted protein was expressed using the standard M9 high-yield growth procedure according to the manufacturer's instructions (Shanghai Medicilon) and then purified as described above, except that 1 mM tris(2-carboxyethyl)phosphine was added to all buffers. All crystals were grown using the sitting drop method at 22°C. Apoprotein crystals of PoGH74cat were grown using 1.5 µl of 20 mg/ml protein with 10 mM xylose and 0.1 M glucose plus 1.5 µl of the reservoir solution 25% (w/v) PEG 8K, 100 mM Bis–Tris buffer, pH 6.9, and 0.2 M sodium acetate. Crystals of PoGH74cat in complex with XLX were grown by co-crystallization using 0.5 µl of 10 mg/ml protein plus 5 mM of a xyloglucan oligosaccharide mixture (XXXG, XLXG, XXLG, and XLLG) plus 0.5 µl of the reservoir solution 20% (w/v) PEG3350 and 0.2 M sodium potassium tartrate. Crystals of the PoGH74catD70A mutant in complex with XXLG and XGXXLG were obtained by co-crystallization of 0.5 µl of 10 mg/ml protein with a complex XyGO mixture plus 0.5 µl reservoir solution 25% (w/v) PEG3350, 0.2 M magnesium chloride, and 100 mM Tris buffer, pH 8.5. All crystals were cryoprotected using 15% glycerol before flash cooling using a nitrogen stream.

X-ray diffraction data for the PoGH74cat apoenzyme and PoGH74cat–XLX complex were collected at 100 K at the selenium absorption peak at beamline 19-ID of the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory, Argonne, IL, U.S.A. X-ray diffraction for the PoGH74catD70A-XXLG + XGXXLG complex was collected at 100 K at beamline 08-ID at the Canadian Macromolecular Crystallography Facility, Canadian Light Source, Saskatoon, Saskatchewan, Canada. X-ray diffraction data were reduced using HKL-3000 [55] or XDS and Aimless [56,57]. The initial structure of PoGH74catD70A-XXLG + XGXXLG was solved by Molecular Replacement (MR) using PHENIX.phaser [58] and the structure of Xgh74A (PDB 2CN3, [37]). An initial model of the protein was built using PHENIX.autobuild, followed by manual model building and refinement with Coot [59] and PHENIX.refine. The subsequent structures of PoGH74cat + XLX and PoGH74cat (apoenzyme) were solved by MR. The presence of all ligand molecules was readily apparent in Fo − Fc maps after resolving the positions of the protein atoms. All B-factors were refined, and TLS parameterization was included in final rounds of refinement. All geometry was verified using the PHENIX and the wwPDB server, and structures were deposited to the Protein Databank with accession numbers 6MGJ, 6MGK, and 6MGL.

Results and discussion

Primary structure analysis

A BLASTp search showed that mature PoGH74 is composed of an N-terminal GH74 catalytic module in-train with three modules of unknown function (‘X2 domain’, PFAM03442, [60]) and a carbohydrate-binding module (CBM) family 3 (Figure 1A). The GH74 catalytic module (Figure 1B) shares more than 80% identity with a group of 17 uncharacterized proteins belonging to the genus Paenibacillus (data not shown). The closest related characterized GH74 enzymes are Paenibacillus sp. KM21 XEG74 (GenBank BAE44527) [30] and Cellvibrio japonicus CjGH74 (GenBank ACE84745) [17], which are 68% and 58% identical with PoGH74cat, respectively. The amino acid sequence alignment of PoGH74cat with Paenibacillus sp. KM21 XEG74, and all GH74 enzymes that have a defined 3D structure (Figure 1C), revealed conservation of the catalytic residues (Asp70 and Asp477) and the absence of the active-site blocking loop that confers the exo-specificity to Geotrichum sp. GH74 OXG-RCBH [39] and Aspergillus nidulans OREX [18]. Notable sequence differences in PoGH74cat are the loop comprised of Asn642–Ala651, which is exclusive to PoGH74cat, and the loop comprised of Asn671–Asn675. This latter loop is absent in approximately one-half of the GH74 structural representatives and its sequence is not conserved in other 3D structural representatives, namely Paenibacillus sp. KM21 XEG74 (GenBank BAE44527), C. japonicus GH74 (GenBank ACE847445), and a Streptomyces GH74 member (GenBank AEN08502) (Figure 1C).

Modular architecture of the native P. odorifer AIQ73809 gene product.

Figure 1.
Modular architecture of the native P. odorifer AIQ73809 gene product.

(A) The full-length gene product is composed of a signal peptide, a GH74 catalytic domain, three X2 domains, and one family 3 carbohydrate-binding module. (B) Recombinant PoGH74cat produced with a 6×-His tag on the N-terminus. (C) Protein sequence alignment of PoGH74cat with the closest related characterized GH74 enzyme (XEG74) and with all the enzymes that have a 3D structure known in the GH74 family. All proteins are indicated according to their name and their GenBank accession number. Key sequence regions are shown, with gaps indicated by vertical black lines. Triangles correspond to point mutations, red for the catalytic D70, blue for W347 (+3 subsite), W348 (+5 subsite), Y372 (+6 subsite) and W406 (+2 subsite), and yellow for G476 (−1 subsite). The red star indicates the catalytic residue D477. The exo-loop of the oligoxyloglucan-specific xyloglucanase Geotrichum sp. OXG-RCBH (BAC22065) is indicated by a black line, while the loops Asn642–Ala651 specific to PoGH74cat (AIQ73809) and Asn671–Asn675 are underlined in green. Residue numbering is based on PoGH74 full-length protein.

Figure 1.
Modular architecture of the native P. odorifer AIQ73809 gene product.

(A) The full-length gene product is composed of a signal peptide, a GH74 catalytic domain, three X2 domains, and one family 3 carbohydrate-binding module. (B) Recombinant PoGH74cat produced with a 6×-His tag on the N-terminus. (C) Protein sequence alignment of PoGH74cat with the closest related characterized GH74 enzyme (XEG74) and with all the enzymes that have a 3D structure known in the GH74 family. All proteins are indicated according to their name and their GenBank accession number. Key sequence regions are shown, with gaps indicated by vertical black lines. Triangles correspond to point mutations, red for the catalytic D70, blue for W347 (+3 subsite), W348 (+5 subsite), Y372 (+6 subsite) and W406 (+2 subsite), and yellow for G476 (−1 subsite). The red star indicates the catalytic residue D477. The exo-loop of the oligoxyloglucan-specific xyloglucanase Geotrichum sp. OXG-RCBH (BAC22065) is indicated by a black line, while the loops Asn642–Ala651 specific to PoGH74cat (AIQ73809) and Asn671–Asn675 are underlined in green. Residue numbering is based on PoGH74 full-length protein.

Production and biochemical characterization of PoGH74cat

cDNA encoding the catalytic module of the full-length protein was cloned into a pMCSG53 vector without the signal peptide, and the X2 and the CBM3 modules. The resulting recombinant enzyme, appended with an N-terminal 6×-Histidine tag, PoGH74cat (Figure 1B), was successfully produced in E. coli BL21DE3 and purified with a typical yield of 60 mg of protein per liter of cell culture.

Initial tests indicated that PoGH74cat was able to hydrolyze tamarind seed XyG, so the optimum pH and temperature were evaluated using this polysaccharide as a substrate. The recombinant enzyme was active in a range of pH from 5 to 8 with an optimum activity at pH 6.0 in 50 mM sodium phosphate buffer (Supplementary Figure S1A). The highest activity was observed at 50°C (Supplementary Figure S1B), and approximately 70% of the original activity remained after a 24 h incubation at 37°C (Supplementary Figure S2).

Under subsequent standard assay conditions (50 mM sodium phosphate buffer, pH 6.0, 37°C), PoGH74cat showed no endo-mannanase activity towards konjac glucomannan, no endo-xylanase activity towards wheat flour arabinoxylan or beechwood xylan, and no endo-glucanase activity towards CM-cellulose or barley mixed-linkage β(1–3)/β(1–4)-glucan. Low levels of activity were detected using HE-cellulose, which together with the above data indicated a strict specificity towards polysaccharides composed of a β-(1,4)-linked glucosyl backbone. To define further the substrate specificity of PoGH74cat, Michaelis–Menten analysis were performed at 37°C in sodium phosphate buffer, pH 6.0, for XyG and HE-cellulose (Table 1 and Supplementary Figure S3). Comparison of kcat/KM values indicates that PoGH74cat has ∼3000-fold higher specificity for XyG than for HE-cellulose, underscoring the importance of α-(1,6)-xylopyranosyl substitution (KM and kcat values for XyG of 0.05 mg/ml and 39.8 s−1, respectively, and KM and kcat values for HE-cellulose of 3.05 mg/ml and 0.83 s−1, respectively).

Table 1
Michaelis–Menten kinetics of PoGH74cat and variants on tamarind seed XyG
 KM (mg/ml) kcat (s−1kcat/KM 
PoGH74cat 0.05 ± 0.01 39.8 ± 3.8 796 
PoGH74cat(G476Y) (−1 subsite) 0.11 ± 0.02 25.3 ± 1.3 230 
PoGH74cat(W406A) (+2 subsite) 0.17 ± 0.02 43.8 ± 1.5 258 
PoGH74cat(W347A) (+3 subsite) 0.23 ± 0.01 214.7 ± 9.8 930 
PoGH74cat(W348A) (+5 subsite) 0.22 ± 0.01 58.3 ± 4.3 265 
PoGH74cat(Y372A) (+6 subsite) 0.04 ± 0.01 47.5 ± 1.5 1187 
 KM (mg/ml) kcat (s−1kcat/KM 
PoGH74cat 0.05 ± 0.01 39.8 ± 3.8 796 
PoGH74cat(G476Y) (−1 subsite) 0.11 ± 0.02 25.3 ± 1.3 230 
PoGH74cat(W406A) (+2 subsite) 0.17 ± 0.02 43.8 ± 1.5 258 
PoGH74cat(W347A) (+3 subsite) 0.23 ± 0.01 214.7 ± 9.8 930 
PoGH74cat(W348A) (+5 subsite) 0.22 ± 0.01 58.3 ± 4.3 265 
PoGH74cat(Y372A) (+6 subsite) 0.04 ± 0.01 47.5 ± 1.5 1187 

In comparison, previously characterized GH74 enzymes exhibit KM values in the range of 0.1–1 mg/ml and kcat values similar to that of PoGH74cat [17,25,30,33]. These biochemical properties highlight the high specificity of PoGH74cat towards XyG in the context of P. odorifer metabolism. Indeed, this essentially exclusive specificity for XyG is typical for GH74 members [1735], with the exception of Thermotaga maritima Cel74, which has been reported to have a 4-fold higher activity towards barley mixed-linkage β-glucan than tamarind seed XyG [20].

Mode of action of PoGH74cat

The well-characterized Bacteroides ovatus BoGH5 [11] was used as a benchmark for endo-dissociative xyloglucanase activity when investigating the mode of action of PoGH74cat on XyG. Using an equivalent number of activity units, PoGH74cat and BoGH5 released soluble dye fragments from cross-linked AZCL-XyG at similar rates over 2 h (Supplementary Figure S4), thereby indicating that PoGH74cat also acts as an endo-xyloglucanase. Subsequently, measurement of the decrease in XyG viscosity compared with the release of reducing sugars for BoGH5 revealed a rapid reduction in viscosity with increasing polysaccharide hydrolysis, as expected for an endo-dissociative GH (Figure 2). In contrast, the viscosity reduction with PoGH74cat exhibited a more linear profile that is indicative of processivity, i.e. multiple hydrolysis events without polysaccharide chain release.

Normalized flow time of xyloglucan solutions measured in an Ubbelohde viscometer following hydrolysis by PoGH74cat variants.

Figure 2.
Normalized flow time of xyloglucan solutions measured in an Ubbelohde viscometer following hydrolysis by PoGH74cat variants.

Wild-type (red circles), PoGH74cat(W406A) (+2 subsite) (green diamonds), PoGH74cat(W347A) (+3 subsite) (blue triangles), PoGH74cat(W348A) (+5 subsite) (purple triangles), PoGH74cat(Y372A) (+6 subsite) (cyan hexagons), and BoGH5 (black squares).

Figure 2.
Normalized flow time of xyloglucan solutions measured in an Ubbelohde viscometer following hydrolysis by PoGH74cat variants.

Wild-type (red circles), PoGH74cat(W406A) (+2 subsite) (green diamonds), PoGH74cat(W347A) (+3 subsite) (blue triangles), PoGH74cat(W348A) (+5 subsite) (purple triangles), PoGH74cat(Y372A) (+6 subsite) (cyan hexagons), and BoGH5 (black squares).

Commensurate with these quantitative analyses, product analysis by HPAEC-PAD (Figure 3A) and MALDI-TOF MS (Table 2 and Supplementary Figure S5) likewise indicated that PoGH74cat is processive. As previously reported [11], the dissociative endo-xyloglucanase BoGH5 generated a wide distribution of oligo- and polysaccharides at the early stage of the reaction, which was reduced to the canonical set of Glc4-based tamarind XyGOs (i.e. XXXG, XLXG, XXLG, and XLLG) after extended incubation (Supplementary Figure S6). In contrast, PoGH74cat released a range of small XyGOs (corresponding to a backbone length of Glc2 to Glc7) already in early stages of hydrolysis of XyG, which continued to accumulate. XyGOs with a retention times between 9 and 11 min (Figure 3A) have larger than Glc4-based backbones, and MALDI-TOF analysis revealed three main products with m/z values of 1571, 1865, and 2027, potentially corresponding to sodium adducts of Glc5- and Glc6-backbone XyGOs, [GXLLG + Na]+, [XGXLLG + Na]+, and [LGXLLG + Na]+, respectively. Low levels of ions with m/z values of 1703 and 1997 were also observed, potentially corresponding to [LLGXX + Na]+ (Glc5-backbone) and [XLGXXXG + Na]+ (Glc7-backbone), respectively.

Hydrolysis of tamarind seed xyloglucan hydrolysis by PoGH74 variants monitored by HPLC.

Figure 3.
Hydrolysis of tamarind seed xyloglucan hydrolysis by PoGH74 variants monitored by HPLC.

(A) Wild-type PoGH74cat, (B) PoGH74cat(G476Y) (−1 subsite), (C) PoGH74cat(W406A) (+2 subsite), (D) PoGH74cat(Y372A) (+6 subsite), (E) PoGH74cat(W347A) (+3 subsite), and (F) PoGH74cat(W348A) (+5 subsite). The bars above the chromatograms indicate retention times corresponding to the length of the XyGO backbone, Glcn.

Figure 3.
Hydrolysis of tamarind seed xyloglucan hydrolysis by PoGH74 variants monitored by HPLC.

(A) Wild-type PoGH74cat, (B) PoGH74cat(G476Y) (−1 subsite), (C) PoGH74cat(W406A) (+2 subsite), (D) PoGH74cat(Y372A) (+6 subsite), (E) PoGH74cat(W347A) (+3 subsite), and (F) PoGH74cat(W348A) (+5 subsite). The bars above the chromatograms indicate retention times corresponding to the length of the XyGO backbone, Glcn.

Table 2
XyGOs released from tamarind seed XyG after 2 and 72 h of digestion by PoGH74cat and the −1 subsite variant PoGH74cat(G476Y)
XyGO structure Mass [XyGO] (Da) Mass [XyGO + Na]+ (Da) PoGH74cat PoGH74cat(G476Y) 
2 h 72 h 2 h 72 h 
XG 474 497 − − 
XX 606 629 − − 
LG 636 659 − − 
LX or XXG 768 791 − − 
LL or LGX 930 953 − − 
XXXG 1062 1085 ∼ 
XXLG 1224 1247 ∼ 
XLLG 1386 1409 
GXLLG 1548 1571 ∼   
LLGXX 1680 1703 ∼ − − − 
XGXLLG 1842 1865 − − − 
LGXLLG 2004 2027 − − − 
XLGXXXG 1974 1997 ∼ − − − 
XXXGXLLG 2430 2453 − − − 
XXLGXLLG 2592 2615 − − − 
XLLGXLLG 2754 2778 ∼ − − 
XyGO structure Mass [XyGO] (Da) Mass [XyGO + Na]+ (Da) PoGH74cat PoGH74cat(G476Y) 
2 h 72 h 2 h 72 h 
XG 474 497 − − 
XX 606 629 − − 
LG 636 659 − − 
LX or XXG 768 791 − − 
LL or LGX 930 953 − − 
XXXG 1062 1085 ∼ 
XXLG 1224 1247 ∼ 
XLLG 1386 1409 
GXLLG 1548 1571 ∼   
LLGXX 1680 1703 ∼ − − − 
XGXLLG 1842 1865 − − − 
LGXLLG 2004 2027 − − − 
XLGXXXG 1974 1997 ∼ − − − 
XXXGXLLG 2430 2453 − − − 
XXLGXLLG 2592 2615 − − − 
XLLGXLLG 2754 2778 ∼ − − 

The symbols (+), (−) ,and (∼) indicate the presence, the absence, and traces of product in solution, respectively.

As the XyG hydrolysis reaction progressed, both Glc6 XyGOs (XGXLLG and LGXLLG) and the Glc7 XyGO (XLGXXXG) were completely hydrolyzed by PoGH74cat, leaving only Glc2 to Glc5 XyGOs in solution after 72 h of reaction. The peaks at m/z 497 ([XG + Na]+), 629 ([XX + Na]+), and 659 [LG + Na]+) correspond to Glc2-backbone XyGOs, and the peak at m/z 1409 corresponds to the Glc4-backbone XyGO [XLLG + Na]+. Two other products with m/z values of 791 and 953 were detected, suggesting the presence of [LX + Na]+ or [XXG + Na]+ and [LL + Na]+ or [LGX + Na]+, respectively. Notably, traces of GXLLG remained in solution after 72 h ([GXLLG + Na]+, m/z 1571), indicating the recalcitrance of this bis-galactosylated, Glc5-backbone XyGO product.

Considered together, these results indicate that PoGH74cat acts an endo-processive xyloglucanase, like Phanerochaete chrysosporium Xgh74B [29] and Paenibacillus sp. strain KM21 XEG74(CD) [30], and furthermore has the ability to cleave the glycosidic bonds of both branched and unbranched backbone glucosyl units. Across GH families, most endo-xyloglucanases, like BoGH5, release XXXG-type XyGOs by exclusive hydrolysis at unbranched glucosyl residues, including members of GH74 [17,23,33,37]. However, some other members of GH74 share the slack regiospecificity of PoGH74cat, for example, Trichoderma reesei Cel74A [22], Xanthomonas citri XGHA [24], and Streptomyces avermitilis GH74A and GH74B [27]. Notably, the apparently limited ability of PoGH74cat to hydrolyze XLLG, XGXLL, XGXLLG, and LGXLLG suggests that bis-galactosylation significantly hinders catalysis.

Structural analysis of xyloglucan recognition by PoGH74cat

To elucidate the structural basis for PoGH74cat bond cleavage pattern and the mode of action, we solved the tertiary structure of the catalytic module in multiple substrate-bound states. We crystallized the free (‘apo’) wild-type enzyme, a wild-type complex with the xyloglucan fragment XLX, and the inactive D70A catalytic base mutant in complex with two xyloglucan fragments (XXLG and XGXXLG). In particular, this last structure represents the most extensive GH74–ligand complex solved to-date [17,36,37,39,61]. We determined the initial apo structure of PoGH74cat from a selenomethione-derivatized protein crystal using Molecular Replacement and the structure of Xgh74A [37]. Subsequent structures were determined using Molecular Replacement and the apo structure. All X-ray crystallographic statistics are shown in Table 3.

Table 3
X-ray crystallographic statistics
Structure PoGH74cat PoGH74cat·XLX PoGH74catD70A·(XXLG + XGXXLG) 
PDB code 6MGJ 6MGK 6MGL 
Data collection 
Space group CCP212121 
Unit cell 
  a, b, c (Å) 239.1, 226.7, 187.2 239.3, 212.5, 91.6 61.2, 100.8, 118.7 
 α, β, γ (°) 90, 114.1, 90 90, 105.5, 90 90, 90, 90 
Resolution, Å 40.0–2.00 40.0–2.10 46.41–1.50 
 Rmerge1 0.125 (0.704)* 0.093 (0.479) 0.057 (0.455) 
 Rpim2 0.056 (0.315) 0.055 (0.313) 0.031 (0.352) 
CC1/2 0.929 0.932 0.701 
 I/σ(I12.33 (2.0) 11.4 (2.2) 14.5 (2.0) 
Completeness, % 99.7 (99.7) 99.0 (97.1) 99.5 (94.9) 
Redundancy 5.4 (5.5) 3.6 (3.1) 4.0 (3.1) 
Refinement 
Resolution, Å 39.78–2.00 39.29–2.10 46.41–1.50 
No. unique reflections: working, test 601755, 3538 236912, 21297 117296, 5836 
 R-factor/free R-factor3 14.9/17.7 (22.2/27.0) 15.8/18.1 (21.7/25.0) 14.8/17.7 (25.1/27.2) 
No. refined atoms, molecules 
 Protein 45387, 8 22671, 4 5730, 1 
 Ligand NA 228 197 
 Solvent 412 354 35 
 Water 9781 3470 1491 
B-factors 
 Protein 28.00 29.1 18.3 
 Ligand NA 36.0 26.6 
 Solvent 72.6 71.4 57.0 
 Water 43.8 41.3 36.7 
r.m.s.d. 
 Bond lengths, Å 0.004 0.003 0.008 
 Bond angles, ° 0.741 0.693 1.098 
Structure PoGH74cat PoGH74cat·XLX PoGH74catD70A·(XXLG + XGXXLG) 
PDB code 6MGJ 6MGK 6MGL 
Data collection 
Space group CCP212121 
Unit cell 
  a, b, c (Å) 239.1, 226.7, 187.2 239.3, 212.5, 91.6 61.2, 100.8, 118.7 
 α, β, γ (°) 90, 114.1, 90 90, 105.5, 90 90, 90, 90 
Resolution, Å 40.0–2.00 40.0–2.10 46.41–1.50 
 Rmerge1 0.125 (0.704)* 0.093 (0.479) 0.057 (0.455) 
 Rpim2 0.056 (0.315) 0.055 (0.313) 0.031 (0.352) 
CC1/2 0.929 0.932 0.701 
 I/σ(I12.33 (2.0) 11.4 (2.2) 14.5 (2.0) 
Completeness, % 99.7 (99.7) 99.0 (97.1) 99.5 (94.9) 
Redundancy 5.4 (5.5) 3.6 (3.1) 4.0 (3.1) 
Refinement 
Resolution, Å 39.78–2.00 39.29–2.10 46.41–1.50 
No. unique reflections: working, test 601755, 3538 236912, 21297 117296, 5836 
 R-factor/free R-factor3 14.9/17.7 (22.2/27.0) 15.8/18.1 (21.7/25.0) 14.8/17.7 (25.1/27.2) 
No. refined atoms, molecules 
 Protein 45387, 8 22671, 4 5730, 1 
 Ligand NA 228 197 
 Solvent 412 354 35 
 Water 9781 3470 1491 
B-factors 
 Protein 28.00 29.1 18.3 
 Ligand NA 36.0 26.6 
 Solvent 72.6 71.4 57.0 
 Water 43.8 41.3 36.7 
r.m.s.d. 
 Bond lengths, Å 0.004 0.003 0.008 
 Bond angles, ° 0.741 0.693 1.098 
*

Values in brackets refer to highest resolution shells.

1

Rmerge = ΣhklΣj|Ihkl.j − 〈Ihkl〉|/ΣhklΣjIhk,j, where Ihkl,j and 〈Ihkl〉 are the jth and mean measurement of the intensity of reflection j.

2

Rpim = Σhkl√(n/n − 1) Σnj = 1|Ihkl.j − 〈Ihkl〉|/ΣhklΣjIhk,j..

3

R = Σ|Fpobs − Fpcalc|/ΣFpobs, where Fpobs and Fpcalc are the observed and calculated structure factor amplitudes, respectively.

Overall, PoGH74cat is structurally similar to previously determined GH74 crystal structures, which comprise two 7-bladed β-propeller domains (Figure 4A and Supplementary Figure S7). The juxtaposition of the two domains forms a long and wide cleft (greater than 50 × 17 × 13 Å) with the catalytic residues D70 and D477 positioned its center (Figure 4B). This cleft is lined with aromatic amino acids including eight tryptophan (W96, W125, W323, W347, W348, W367, W369, and W406) and four tyrosine residues (Y121, Y213, Y294, and Y372), forming a large hydrophobic platform that extends from the −4 to the +6 subsite (nomenclature according to [62]).

Structural analysis of xyloglucan binding to PoGH74cat.

Figure 4.
Structural analysis of xyloglucan binding to PoGH74cat.

(A) Overlay of crystal structures of PoGH74cat apoenzyme, PoGH74cat·XLX, and PoGH74cat(D70A)·(XXLG + XGXXLG) complexes; protein backbones are shown in gray cartoon with ligands colored magenta and pink. −4 and +6 subsites representing the extreme ends of the bound ligands are labeled. Mobile loop 642–651 and its partner loop 671–675 are colored in green from the PoGH74cat(D70A)·(XXLG + XGXXLG) complex crystal structure. Catalytic residues D70 and D477 are colored red. (B) Surface representation of PoGH74cat(D70A)·(XXLG + XGXXLG) complex structure, with mobile loop 642–651 colored green, residues A70 and D477 colored red, and aromatic residues constituting the positive subsites W406 (subsite +2), W347 (subsite +3), W348 (subsite +5), and Y372 (subsite +6) colored in blue. (C) Details of PoGH74cat·XLX structure, with active site residues shown in sticks and ligand in ball-and-stick. −1, −2 and −3 subsites labeled. Electron density shown is Fo − Fc map calculated without ligand co-ordinates contoured at 3.0 σ. (D) Details of PoGH74cat(D70A)·(XXLG + XGXXLG) structure, with active-site residues shown in sticks and ligand in ball-and-stick. −4 through +6 subsites labeled. Electron density shown is Fo − Fc map calculated without ligand co-ordinates contoured at 3.0 σ.

Figure 4.
Structural analysis of xyloglucan binding to PoGH74cat.

(A) Overlay of crystal structures of PoGH74cat apoenzyme, PoGH74cat·XLX, and PoGH74cat(D70A)·(XXLG + XGXXLG) complexes; protein backbones are shown in gray cartoon with ligands colored magenta and pink. −4 and +6 subsites representing the extreme ends of the bound ligands are labeled. Mobile loop 642–651 and its partner loop 671–675 are colored in green from the PoGH74cat(D70A)·(XXLG + XGXXLG) complex crystal structure. Catalytic residues D70 and D477 are colored red. (B) Surface representation of PoGH74cat(D70A)·(XXLG + XGXXLG) complex structure, with mobile loop 642–651 colored green, residues A70 and D477 colored red, and aromatic residues constituting the positive subsites W406 (subsite +2), W347 (subsite +3), W348 (subsite +5), and Y372 (subsite +6) colored in blue. (C) Details of PoGH74cat·XLX structure, with active site residues shown in sticks and ligand in ball-and-stick. −1, −2 and −3 subsites labeled. Electron density shown is Fo − Fc map calculated without ligand co-ordinates contoured at 3.0 σ. (D) Details of PoGH74cat(D70A)·(XXLG + XGXXLG) structure, with active-site residues shown in sticks and ligand in ball-and-stick. −4 through +6 subsites labeled. Electron density shown is Fo − Fc map calculated without ligand co-ordinates contoured at 3.0 σ.

The electron density for XLX as bound to the wild-type enzyme and XXLG + XGXXLG as bound to the D70A mutant were unambiguous, allowing for validation that the central cavity is the substrate-binding cleft (Figure 4C,D). XLX was observed to bind in the negative subsites (Figure 4C). This compound formed stacking interactions with W96, W125, and Y213, respectively. XLX also formed numerous hydrogen-bonding interactions with surrounding residues. In particular, the O1 and O2 atoms of the reducing-end glucosyl residue formed interactions with the catalytic D477 and D70 residues, respectively. In the crystal structure of the PoGH74cat(D70A) mutant bound to XXLG and XGXXLG, XXLG bound in the −4 through −1 subsite, with the XLG component binding nearly exactly as XLX was bound to the wild-type enzyme (Figure 4D). XGXXLG was primarily recognized via stacking interactions; subsites +1 through +6 were defined through stacking interactions with the XGXXLG ligand via Y294, W406, W347, W348, W323, and Y372 (Figure 4D). Notably, the subsite +6 in PoGH74cat is unique among GH74 members.

A comparison of the protein backbone in these three crystal structures revealed a conformational change of the loop between residues 642–651, which contributes to the −4 subsite (Figure 4A,B). The rear surface of this loop packs against a second loop (671–675). Notably, the loop comprising Asn642–Ala651 is unique to PoGH74 [e.g. it is absent in the exemplar endo-dissociative xyloglucanase XEG74 from Geotrichum sp. (PDB ID 3AOF, Supplementary Figure S7, and Figure 1C)], while the loop Asn671–Asn675 has limited distribution among GH74 members (Figure 1C).

Structural basis of the bond cleavage pattern of PoGH74cat

In GH74, deletion of the exo-loop located past the +2 subsite of the Geotrichum sp. reducing-end-specific cellobiohydrolase (OXG-RCBH) switched the cleavage pattern of the enzyme from a strict exo-acting enzyme to an endo-xyloglucanase [38]. Similarly, we hypothesized that loop Asn642–Ala651, which protrudes into the active-site cleft near subsite −4, and the second-shell loop Asn671–Asn675, could confer exo-like activity to PoGH74cat. Consequently, both loops were individually deleted and the variants ΔAsn642–Ala651 and ΔAsn671–Asn675 were successfully produced in E. coli BL21 DE3. Michaelis–Menten kinetic analysis and the identification of the hydrolysis products using XyG as a substrate showed that they both behaved like the wild-type enzyme (data not shown). Thus, although Asn642–Ala651 is the first mobile active-site loop to be observed in GH74, its function remains enigmatic, similar to examples in other families (e.g. GH3 and GH11 [63,64]).

We then turned our attention to the role of the Gly-476 residue near the −1 subsite of PoGH74cat, which is conserved in most GH74 enzymes. However, a tyrosine occupies this position in the Geotrichum sp. XEG (Figure 1C and Supplementary Figure S7), the mutation of which to glycine has been shown to relax the XyG backbone regiospecificity of this enzyme [38]. The PoGH74cat(G476Y) variant was successfully produced in E. coli BL21 DE3 and Michaelis–Menten analysis showed that the KM of the variant was 2-fold higher and the overall catalytic efficiency was ∼4-fold lower compared with the wild-type on XyG (Table 1). Like the wild type, PoGH74cat(G476Y) first generated small XyGOs during the initial stages of polysaccharide hydrolysis (Figure 3B). Strikingly, however, the variant produced only Glc4-backbone (XXXG-type) XyGOs along with higher order congeners. MALDI-TOF MS analysis confirmed the presence of XXXG, XXLG, XLXG, and XLLG, along with three putative Glc8-backbone XyGOs: XLLGXXXG, XLLGXXLG, and XLLGXLLG (Table 2 and Supplementary Figure S5B). As the reaction progressed, the Glc8-backbone XyGOs were fully degraded into Glc4-backbone XyGOs after 72 h of reaction (Supplementary Figure S5D).

Taken together, these results indicate that the G476Y variant has strict substrate recognition and can onlycleave the backbone of XyG at the unsubstituted glucosyl units. These results directly mirror those of Yaoi et al. for the opposite (Y457G) mutation in Geotrichum sp. XEG (cf. Supplementary Figure S7). However, although the bond cleavage pattern of these enzymes is driven by the nature of a single residue, the structural basis of regiospecificity in GH74 seems to be more complex. Indeed, Paenibacillus sp. KM21 XEG74 [30], C. japonicus CjGH74 [17], and Clostridium thermocellum XGH74A [37] all harbor a glycine residue at the corresponding position, but cleave XyGs specifically at unsubstituted glucosyl units. Future structure–function analyses will be required to fully disentangle the combined effects of other active-site residues in these enzymes.

Structural basis of the processivity of PoGH74cat

Given the remarkably large hydrophobic platform and the unique +6 subsite of the PoGH74cat active cleft, we investigated the key determinants for the processive mode of action of PoGH74cat by targeting the aromatic residues constituting the positive subsites (Figure 4). Overall, only marginal changes in Michaelis–Menten kinetics on XyG were observed for the site-directed mutants W406A (subsite +2), W347A (subsite +3), W348A (subsite +5), and Y372A (subsite +6). The KM values of the variants W406A, W347A, and W348A were increased by 2- to 5-fold compared with the wild-type enzyme, indicating the involvement of these main platform Trp residues in substrate recognition, while the KM value of the Y372A mutant (+6 subsite) was indistinguishable versus the wild-type enzyme. In general, the turnover rates of all variants were similar to the wild-type (less than 2-fold different), with the exception of W347A, the kcat value of which was increased 5-fold compared with the wild-type enzyme. These combined changes resulted in kcat/KM values that were similar to, or slightly reduced from, the wild-type value (Table 1).

Substrate conversion-dependent viscosity measurements more clearly delineated the catalytic properties of the enzyme variants (Figure 2). The W406A (+2 subsite) and Y372A (+6 subsite) variants caused a more rapid decrease in the specific viscosity as a function of hydrolysis degree than the wild type. This effect was even more pronounced for the W347A (+3 subsite) and W348A (+5 subsite) variants, which directly recapitulated the behavior of the canonical dissociative endo-xyloglucanase BoGH5. Product analysis by HPAEC-PAD reflected these shifts in mode of action for all variants. Like the wild-type, the W406 and Y372A variants released smaller size XyGOs but with a distinct tendency to produce longer XyGOs at initial stages of the reaction than the wild type (Figure 3C,D, cf. Figure 3A). In contrast, the W347A and W348A variants, like BoGH5 (Figure 3B), produced large polysaccharide fragments at the early stages of the reactions, which were eventually reduced by random attack to XyGOs (Figure 3E,F).

Taken together, these results indicate that W406 (+2 subsite), W347 (+3 subsite), W348 (+5 subsite), and Y372 (+6 subsite) all contribute to the processivity of PoGH74cat but to different degrees. W347 and W348 in the central region of the positive subsites are strictly essential for processivity. On the other hand, individual removal of W406 and Y372 reduces processivity but does not abolish it (Figures 2 and 3), which suggests that both of these residues synergize with W347 and W348 in the wild-type enzyme. The essentiality of the large aromatic sidechains in the +3 and +5 subsites directly recapitulates previous results of Matsuzawa et al. on Paenibacillus sp. KM21 XEG74 and, moreover, indicates that the direction of processivity after initial attack is towards the reducing-end of the xyloglucan chain [30]. Not least, this pair of tryptophan residues is conserved within all previously characterized processive GH74 enzymes and therefore appears to be a hallmark of processivity in this family (the Trp in subsite +2 is conserved among all GH74 members). Notably, the exemplar endo-dissociative xyloglucanase XEG74 from Geotrichum sp. lacks aromatic amino acids altogether in positions corresponding to the +3 to +5 subsites of PoGH74 (Supplementary Figure S7), concordant with the distinct modes of action of these enzymes. At the same time, our identification of an additional +6 subsite in PoGH74 indicates that some enzymes in the family have further extended this core platform with additional benefits to retention of the polysaccharide chain in the active-site cleft. By delineating the active-site residues responsible for the regiospecificity of bond cleavage and processivity of PoGH74, the structural enzymology described here will inform future bioinformatics, enzyme engineering, and applications development of GH74 members in the context of biomass utilization.

Abbreviations

     
  • CAZy

    carbohydrate-active enzyme

  •  
  • CBM

    carbohydrate-binding module

  •  
  • GH

    glycoside hydrolase

  •  
  • HPAEC-PAD

    high-performance anion-exchange chromatography coupled with pulsed amperometric detection

  •  
  • LIC

    ligation-independent cloning

  •  
  • MALDI-TOF MS

    matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometer

  •  
  • MR

    molecular replacement

  •  
  • XyG

    xyloglucan

  •  
  • XyGO

    xyloglucan oligosaccharide

Acknowledgments

We thank Boguslaw Nocek (Structural Biology Center, Advanced Photon Source) and Nobuhiko Watanabe (University of Calgary) for X-ray diffraction data collection. G.A. and H.B. thank Bernard Henrissat (AFMB-CNRS, Aix-Marseille Université) for informative discussions on X2 modularity. This work was supported by NSERC (via a Discovery Grant to H.B. and a Strategic Partnership Grant for Networks to H.B. and A.S. for the Industrial Biocatalysis Network, http://www.ibnet.ca/), the Canada Foundation for Innovation, and the British Columbia Knowledge Development Fund. Waters Corporation is gratefully acknowledged for the provision of the intact protein LC–MS system used in the present study.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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