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The large (1767 amino acid) endo-α-N-acetylgalactosaminidase from Streptococcus pneumoniae (SpGH101) specifically removes an O-linked disaccharide Gal-β-1,3-GalNAc-α from glycoproteins. While the enzyme from natural sources has been used as a reagent for many years, very few mechanistic studies have been performed. Using the recently solved 3-dimensional structure of the recombinant protein as a background, we report here a mechanistic investigation of the SpGH101 retaining α-glycoside hydrolase using a combination of synthetic and natural substrates. Based on a model of the substrate complex of SpGH101 we propose D764 and E796 as the nucleophile and general acid/base residues respectively. These roles were confirmed by kinetic and mechanistic analysis of mutants at those positions using synthetic substrates and anion rescue experiments. pKa values of 5.3 and 7.2 were assigned to D764 and E796 on the basis of the pKa values derived from the bell-shaped dependence of kcat/Km upon pH. The enzyme contains several putative carbohydrate binding modules whose glycan binding specificities were probed using the printed glycan array of the Consortium for Functional Glycomics using the inactive D764A and D764F mutants that had been labeled with Alexfluor 488. These studies revealed binding to galacto-N-biose, consistent with a role for these domains in localizing the enzyme near its substrates.
S. pneumoniae is a gram-positive organism that colonizes the respiratory tract of humans as a commensal organism but can become a pathogen that causes pneumonia, meningitis, bacteraemia and otitis media with significant morbidity and mortality, especially in children (1). Because S. pneumoniae is considered a leading cause of death worldwide, a great deal of research has been directed to determining the factors that contribute to the virulence of this organism. Of major importance to this pathogen is the ability to degrade host glycoproteins, and to metabolize the resultant carbohydrates (2). Consistent with this, S. pneumoniae has a large number of glycoside hydrolases, with 21 CAZY glycoside hydrolase families represented by at least one gene in the sequenced reference strains (3). These hydrolases are both secreted and cell-associated. Indeed the genome sequences of the reference strain S. pneumoniae R6 and of a clinical isolate contain at least 6 cell surface-localized enzymes that are thought to play a role in virulence. (4) (5) (6). These cell-associated enzymes include neuraminidases, hyaluronidases, β-galactosidases, and endo-β-N-acetylglucosaminidases, which have been studied in some detail (reviewed in (7)). A new member of this cell surface family of enzymes belonging to the GH101 family of glycoside hydrolases, endo-β-N-acetylgalactosaminidase (SpGH101), specifically cleaves Gal-β-1,3-GalNAc-α-Ser/Thr (T-antigen, galacto-N-biose), which is the core 1 type O-linked glycan common to mucin glycoproteins. This CAZY GH101 family (3) currently contains proteins from 12 species of bacteria, most of which are commensal human bacteria, though some may also be human pathogens. The S. pneumoniae enzyme has been shown recently to be non-essential, but is involved in aiding adherence of Streptococcus to human airway epithelial cells (8). The substrate specificity of this enzyme, SpGH101, has been studied in some detail with material purified from Streptococcus culture supernatants (9)(10). A related enzyme from Bifidobacterium longum was expressed as a recombinant protein, shown to be a retaining glycoside hydrolase, and candidate catalytic residues suggested through kinetic analyses of mutants of conserved carboxylic amino acids (Glu and Asp) (11). However the catalytic machinery was not positively identified. The recently solved 3D structure of SpGH101 (12), in conjunction with sequence alignments, reveals that the GH101 enzymes are multi-modular proteins with a catalytic domain as well as domains that resemble carbohydrate binding modules (CBMs). These include a CBM32 module, along with degenerate CBM4 and CBM16-like folds, plus modules that resemble a degenerate legume lectin fold. The catalytic domain is a distorted (β/α)8 barrel flanked by a domain of all β-sheet structure: very analogous to the arrangement seen in GH13 α-amylases. The flanking domain likely serves to shield the active site from water during catalysis (13).
Sequence similarity between these GH101 enzymes is highest in the catalytic domain, with many of the absolutely conserved residues being in the active site pocket of this GH domain (Figure 1). Based on the active site structure of the α-amylase from Thermoactinomyces vulgaris R-47 complexed with substrate, we modeled a substrate complex for SpGH101 (12). Earlier kinetic analyses of mutants made at the sites of conserved Glu and Asp residues of the BlGH101 enzyme had suggested two conserved residues to be important for enzyme activity, BlGH101 D682 and BlGH101 D789, which correspond to SpGH101 D658 and SpGH101 D764. However inspection of the active site geometry of these acidic residues in the SpGH101 structure with the substrate modeled into place, based on the α-amylase structure, suggested that the likely catalytic residues are instead D764 (nucleophile) and E796 (acid/base). More recently the three-dimensional structure of BlGH101 has been solved and mutation of E822 (E796 in SpGH101) confirms this as an important catalytic residue, though no detailed studies were performed, simply assays (14).
There is considerable interest in enzymes of this type, both as reagents for removal of O-linked glycans and potentially also for de-novo glycosylation of proteins when used in transglycosylation mode, possibly in engineered versions. However, only limited functional information on enzymes from this family is available. In order to correct this deficiency we have performed a detailed mechanistic characterization of this enzyme to identify the catalytic residues. We have also probed the binding specificities of the putative carbohydrate binding modules by use of a catalytically impaired mutant to examine binding to a carbohydrate microarray containing > 400 carbohydrate structures, many of which would be found in a human host.
Streptococcus pneumoniae R6 was obtained from the ATCC (ATCC # BAA-255) and was grown on Columbia Blood Agar at 37 °C and 5 % CO2. E. coli AD202 (CGSC # 7297 or Ref 3) was used for expression of cloned enzymes. Recombinant E. coli strains were grown in 2YT broth with 150 μg/mL ampicillin at 37 °C for 1.5 hours then induced with 0.5 μM isopropyl 1-thio-β-D-galactopyranoside and grown at 30 °C for 7 hours. The plasmid used for cloning was a modified pCWori+ (15).
All DNA isolations, restriction enzyme digestions, ligations, and transformations were performed as recommended by the supplier. Enzymes were obtained from New England Biolabs (Mississauga, ON). Genomic DNA was isolated using a DNeasy Tissue kit (Qiagen Inc., Mississauga, Ontario). PCR was performed using Phusion polymerase and the program: 94 °C for 5 min, 30 cycles of 94 °C for 30 sec, 55 °C for 30 sec and 72 °C for 120 sec, and finally 72 °C for 10 min. The gene was originally amplified from genomic DNA based on the sequence Sp0328 from S. pneumoniae R6 with primers to remove a 40 amino acid signal sequence at the N-terminal end of the gene. The primers for this amplification and other sequence truncations are shown in Table 1.
DNA was purified using either Qiaquick or Minelute kits from Qiagen Inc (Mississauga, Ontario). Genes digested with NdeI and SalI were ligated into pCW and then used to transform E. coli AD202 by electroporation. Plasmids were isolated using a High Pure Plasmid Isolation kit (Roche Diagnostics, Laval, Quebec). DNA sequencing was performed using an Applied Biosystems (Montreal, Quebec) model 3100 automated DNA sequencer and the manufacturer’s cycle sequencing kit.
Recombinant cultures grown in the presence of IPTG were lysed with an Avestin C5 Emulsiflex cell disrupter (Avestin Ottawa, Ontario) and centrifuged first at 27,000 x g to pellet unbroken cells, and then at 100,000 x g to pellet insoluble material. SpGH101 was partially purified using DEAE-Sepharose chromatography (GE Healthcare, Piscataway, NJ) with a gradient of 20 mM – 1 M NH4OAc pH 7.5. The enzyme was concentrated using Amicon Ultra-15 filtration devices (Millipore, Billerica, MA) and then applied to a Superdex-200 size exclusion column in 10 mM NH4OAc pH 7.5. The enzyme in the pooled fractions was finally applied to a Phenyl-Sepharose column (GE Healthcare, Piscataway, NJ) with a gradient of 1.5 M – 10 mM NH4OAc, pH 7.5. Purity was assessed by SDS-PAGE analysis and protein concentrations were determined using a BCA assay (Pierce Biotechnology Inc., Rockford, IL).
DNP-GalNAc (the α anomer) was synthesized as previously described (17). Gal-β-1,3-GalNAc-α-DNP (DNP-TAg) was synthesized enzymatically from GalNAc-α-DNP using the Campylobacter jejuni CgtB galactosyltransferase as described previously (18). The disaccharide product was purified by chromatography on a Superdex Peptide 30 column (GE Healthcare, 1.5 × 80 cm) which had been equilibrated in 100 mM NH4HCO3. The peptide substrate FCHASE-[T-Ag]-IFNα2b was also synthesized as described previously (18). Purity of the products was determined by capillary electrophoresis on a PACE MDQ system as previously described (19).
A PDB file for the T-antigen disaccharide ligand was made using the SWEET modeling program (20). The ligand was fitted to the SpGH101 PDB entry 3ECQ structure using the PatchDock server (21) and figures were generated using PyMOL v1.1 (22).
Activity was measured using the substrates Gal-β-1,3-GalNAc-α-pNP (pNP-TAg), obtained from Toronto Research Chemicals), Gal-β-1,3-GalNAc-α-DNP (DNP-TAg), or DNP-GalNAc. Assays were performed at 37 °C in 50 mM citrate-phosphate buffer pH 6.5 with 0.1 mg/mL acetylated bovine serum albumin. Assays with pNP-TAg were performed in stopped mode with an equal volume of 0.2 M sodium carbonate, 0.5% sodium dodecylsulphate and measured at 400 nm using an extinction coefficient of 14,000 cm-1M-1. Assays with the DNP-TAg and DNP-GalNAc were performed in continuous mode. For wild type SpGH101 enzyme kinetics, 10 μM – 200 μM of DNP-TAg was incubated followed by the addition of 0.2 nM enzyme. For SpGH101 mutants, 1 μM – 10 μM of DNP-TAg was incubated followed by the addition of 2 nM enzyme. Release of the dinitrophenolate ion was followed at 400 nm using a Varian Cary AV-4000 and rates calculated using an extinction coefficient 10,800 cm−1M−1. Kinetic data were analysed using Grafit 5.0 software. For the chemical rescue experiments, 10 mM – 1 M sodium acetate, sodium formate, sodium azide, sodium acetate or potassium fluoride was added and the pH was checked to ensure it remained at 6.5.
A reaction mixture containing 3.5 mM DNP-TAg, 1 M sodium azide and 5 nM of SpGH101 E796Q mutant was prepared and left at room temperature overnight. Product analysis was performed by thin layer chromatography on 60 F254 silica gel aluminum plates (Merck) run in 7:2:1 (v/v/v) ethyl acetate/methanol/water and developed with 10% ammonium molybdate in 2 M H2SO4 followed by charring. Purification of the final product was performed by directly loading the crude reaction mixture onto a flash column chromatography with Silicagel 60. The NMR spectra were recorded using the Bruker AV-300 MHz spectrometer. High resolution mass spectral data were collected by the mass spectrometry laboratory at the University of British Columbia.
The pH dependence of kcat/KM for SpGH101 was measured using the substrate depletion method at low substrate concentrations ([S] KM, (23). Assays were performed with 50 μM DNP-GalNAc or 5 μM DNP-TAg and the following buffers: 50 mM citrate-phosphate pH 4.4 – 7.0, 50 mM sodium phosphate pH 7.0 – 8.0, 50 mM sodium borate pH 8.0 – 9.0. Absorbance was measured using a Varian Cary AV-4000 and data fitted to a first order curve using Grafit 5.0 software. The kcat/KM values were obtained from these fits by dividing by the enzyme concentration.
SpGH101 D764A, and D764F (1 mg) were labeled with Alexafluor 488 according to the instructions supplied with the dye (Invitrogen). The labeled protein was purified from the un-reacted dye by chromatography on Sephadex G-25 equilibrated in PBS buffer pH 7.4. The labeled protein was analyzed for binding to the printed glycan array from the Consortium for Functional Glycomics version 3.1(D764A) or 4.0 (D764F), by the CORE H facility of the CFG. The protocols are available online at http://www.functionalglycomics.org/glycomics. Protocols cfgPTC_243, and cfgPTC_248 were used to analyze SpGH101 on the array. Binding was examined on arrays with both 100 μM and 10 μM glycan spot density for array version 3.1 with D764A, and at 2 protein concentrations of D764F for the version 4 array.
The gene from S. pneumoniae R6 was amplified from genomic DNA using primers designed to eliminate the signal sequence by starting at amino acid 40 (Figure 2). Attempts were made to prepare shorter versions by truncating the protein 200 amino acids from either the N- or C-terminal ends. The 200 residue N-terminal truncation had only 6.6% of the specific activity of the full length protein (data not shown), and therefore was not examined further. The 200 residue C-terminal truncation, SpGH101 aa40–1567, however, maintained the specific activity of the full length enzyme, but as we were interested in the C-terminal CBM-like domain we chose to work with the SpGH101 aa 40–1767. The enzyme was purified to greater than 90% purity in a three step purification procedure with an 80-fold enrichment in specific activity. Catalytic site mutants were grown and purified as described for the WT enzyme. No significant differences in expression or purification were observed with these mutants.
Kinetic parameters for both DNP-GalNAc and DNP-TAg were measured and are shown in Table 2. The specificity of the enzyme for the T-Ag disaccharide was shown by the 26,500-fold greater kcat/Km for the disaccharide substrate than the monosaccharide. This large difference in specificity is due not only to an increase in affinity, as seen by the 70-fold decrease in Km, but also to a 400-fold increase in kcat. DNP-TAg is therefore a vastly superior substrate for use in mechanistic studies. The 2,4-dinitrophenyl glycoside was chosen for the majority of the kinetic analyses since the greater leaving group ability of DNP makes this a better substrate than the p-nitrophenyl analog. In addition the low pKa (4.0) of DNP ensures that the extinction coefficient of 2,4-DNP does not change significantly with pH above pH 5 and that assay is much more sensitive than with the p-nitrophenyl substrate at pH 6.5, which is the optimal pH for the -SpGH101. The enzyme was also shown by NMR analysis of the reaction progress to act with retention of anomeric stereochemistry (data not shown) similar to the B. longum enzyme BlGH101(11).
Based on the active site substrate complex model the mutants D658A, D764A, and E796A/Q were constructed and analysed using the disaccharide DNP-TAg substrate. The least active of these mutants is D764A, with a kcat/Km value some 700 times lower than that of the wild type enzyme. In fact this is likely a maximum estimate of the activity since, based upon the similarity of its Km value to that of the wild type enzyme, it is quite probable that even this low activity derives from a small amount of contaminating wild type enzyme. The next most active is the D658A mutant, with a kcat/Km that is 16-fold lower than that of the wild type enzyme. However it did not show saturation kinetics with the DNP-TAg substrate, thus individual kinetic constants kcat and Km, could not be determined for this mutant. The most active were the E796A and Q mutants, with kcat/Km values essentially the same as the wild type enzyme. Importantly however the kcat values are about 30 fold lower than that of the wild type enzyme, with the Km values also correspondingly lowered.
The monosaccharide substrate DNP-GalNAc was cleaved only by the WT enzyme and the E796A mutant relatively efficiently, but at an extremely low rate for the E796Q mutant. Notably the kcat value for this substrate with E796A was 3 fold higher than for the WT enzyme, though the Km values were similar. However the kcat value was some 300–400 - fold lower than that of the disaccharide substrate, and the Km value some 80-fold higher. These high Km values are suggestive of glycosylation step being rate-limiting. Interestingly a parallel situation was seen with the xylanase Cex from Cellulomonas fimi, where the “addition” of a glucose residue to a monosaccharide substrate was seen to change the rate-limiting step from glycosylation to deglycosylation, and a rationale for this was developed. (24) (25)
In order to investigate the enzymatic activity of the active site mutants on a more natural substrate, a synthetic glycopeptide, FCHASE-[Tag]-IFNα2b was prepared as previously described (18). This substrate contains a T-Antigen disaccharide attached to the threonine side chain of a seven-amino-acid peptide via an α-glycosidic linkage, with a fluorescent label on the N-terminus. Upon the addition of 34 nM of WT SpGH101, 0.5 mM of FCHASE-[TAg]-IFNα2b was rapidly cleaved, with hydrolysis being complete within three hours at room temperature, (data not shown). However, the addition of either 290 nM of E796A or 220 nM of E796Q to 0.5 mM of FCHASE-[TAg]-IFNα2b, resulted in no observable cleavage, even after incubation for two days.
To investigate the roles of D764 and E796 in catalysis, assays were performed in the presence of the exogenous anionic nucleophiles (Table 3). In the case of the proposed acid/base (E796) mutants, both azide and formate increased the steady state activity in a dose-dependent manner, with the greatest increase being seen with azide and the E796Q mutant. Azide rescue was also observed with the E796A mutant, but to a lesser extent. The only anion that restored activity to the nucleophile mutant (D764A), was azide, which has the highest nucleophilicity of those tested (azide, formate, acetate and fluoride). In order to rule out the possibility of these changes in kcat and Km simply resulting from salt effects, the kinetic parameters of the E796 mutants in the presence of either 100 mM or 500 mM NaCl were measured. However, in none of the cases were the Km values of the mutants affected by the addition of Cl−. Anion rescue experiments with the E796A/Q mutants were also attempted using the monosaccharide substrate DNP-GalNAc, however, no anion rescue was observed, consistent with the conclusion that the glycosylation step is rate-limiting for the monosaccharide substrates.
TLC analysis of the reaction mixture of DNP-TAg, sodium azide and SpGH101 E796Q mutant revealed a new non-UV active spot (Rf = 0.32), which is distinct from DNP-TAg (Rf = 0.49) and TAg (Rf = 0.14). After purification, high resolution mass spectra confirmed that the new product has the molecular formula for the TAg-azide. (Calcd. for C14H24N4O10+Na+: 431.1390, Found: 431.1393). The α configuration of the anomeric azide was clearly demonstrated by the small J1,2 coupling constant of 4.2 Hz from the 1H-NMR of the purified product. The relatively big chemical shift (δ = 5.52 ppm) is also very typical for α-glycosyl azide since a much lower chemical shift would be expected for a β glycosyl azide. 1H-NMR (300 MHz, CD3OD) δ: 5.52 (1H, d, J1,2 = 4.2, H-1), 4.45 (1H, dd, J2,3 = 11.2 Hz, J1,2 = 4.2 Hz, H-2), 4.41 (1H, d, J1′,2′ = 7.5 Hz, H-1′), 4.20 – 3.43 (m, 11H), 1.89 (3H, s, COCH3).
In order to probe the carbohydrate binding properties of the SpGH101 enzyme we used the nucleophile mutant as probe on a glycan microarray. The strategy was to use two versions of this mutation, D764A in which the enzyme could potentially bind through the active site, but would not hydrolyze glycans on the array, and D764F which would block binding through the active site by the presence of the bulky Phe residue. In the latter mutant only CBM type interactions should mediate binding to the glycan array. The binding profile revealed strong binding to the disaccharide that is the natural substrate, as well as some binding to the 3-O-sulphated version of this disaccharide (Figure 4A). There were several other weak interactions which disappear when the glycan array contained only low density carbohydrates (data not shown). The binding profile with the D764F mutant was essentially the same although the total binding strength appeared to be lower.
The kinetic and mechanistic data accumulated provide very strong evidence that D764 acts as the catalytic nucleophile (pKa = 5.3) and that E796 functions as the acid/base catalyst (pKa = 7.2) in the double-displacement mechanism followed by SpGH101 (Fig. 5). These are the roles that were proposed on the basis of modeling of substrate binding into the active site using the structure of an α-amylase/substrate complex as a guide. Strong support for the assignment of D764 as the catalytic nucleophile comes from the observation that mutation to alanine results in essentially complete abrogation of enzyme activity. Even with the most active 2,4-dinitrophenyl T-antigen substrate the activity was some 700-fold lower than that of the wild type enzyme. Indeed, even this activity was most probably due to contaminating wild type enzyme. Such essentially complete loss of activity has been seen in essentially all retaining glycosidases when their catalytic nucleophiles have been mutated (26)
Evidence in support of a role for E796 as the acid/base catalyst is particularly strong. The mutants E796A and Q efficiently cleave substrates containing the highly reactive dinitrophenyl leaving group, which does not need acid catalytic assistance, with kcat/Km values essentially identical to that of the wild type enzyme. By contrast, cleavage of natural, non-activated glycopeptide substrates, which do need acid catalytic assistance, was severely compromised. Indeed no cleavage whatsoever was observed using sensitive fluorescent substrates, even after incubations with almost 10 times as much enzyme and incubating for two days. This contrasting behavior with the two different substrates is what has been seen for other retaining glycosidases in which the acid catalyst is mutated, and is consistent entirely with the role. (27)(28)
A more detailed inspection of the kinetic data for the dinitrophenyl glycoside also reveals very low Km values for the acid mutants. This is not a consequence of improved affinity per se, but rather indicates the accumulation of the glycosyl enzyme intermediate. This arises because the glycosylation step remains fast (as shown by kcat/Km values) as a consequence of the excellent leaving group ability of the dinitrophenyl group, but the deglycosylation step is slowed (as seen in kcat) due to the removal of general base catalysis.
Further, very strong evidence for E796 as the acid/base catalyst derives from the rescue of steady state activity seen in the presence of the anionic nucleophiles azide and formate. Increases in kcat of up to 5 fold were observed, with no effect upon kcat/Km. This arises because azide is a much better nucleophile than water, but cannot attack the glycosyl-enzyme formed on the wild-type enzyme due to electrostatic screening from the deprotonated E796. Removal of that charge in the alanine mutant allows direct attack of azide or formate at the anomeric centre, with associated increases in steady state rate. Parallel studies with sodium chloride confirm that the rate increases are not due to salt effects.
The relatively small increases in steady state rate observed in these anion rescue experiments are consistent with findings on other α-glycosidases (29) (30) where increases of 5–10 fold have been seen. This contrasts with the much larger increases typically seen for catalytic acid mutants of β-glycosidases. (27)(31). The ‘saturation’ behaviour seen in such experiments (Fig. 6) is not a reflection of saturable reversible binding of the anion, but rather is a consequence of a change in rate-limiting step at higher anion concentrations. The smaller increases seen for the α-glycosidases compared to those seen for the β-glycosidases are, then, a reflection of the inherent relative rates of glycosylation and deglycosylation steps in α- and β-glycosidases. This most likely arises primarily from the somewhat greater reactivity of β-glycosidases than their α-anomers. Interestingly, no anion rescue was observed when DNP GalNAc was used as substrate, consistent with the indications (from high Km values) that the glycosylation step, rather than the deglycosylation step, is rate-limiting in that case. Further confirmation of the mechanism shown in Figure 6 is provided by the isolation of the α-configured azide adduct of the T-antigen substrate from such reaction mixtures.
The mechanistic role of D658 is less clear. Structural alignment with α-amylase places this residue on top of Y82, a residue that has been suggested to form hydrophobic interactions with the sugar. Clearly such a role is not possible for D658, both since the residue is not hydrophobic and because the hydroxyl is axial-configured, thereby decoupling the hydrophobic face. Indeed, modeling would suggest the formation of hydrogen bonds between D658 and the axial hydroxyl of the substrate. The large increase in Km value (no saturation was seen) is consistent with a role in substrate binding, as is the 15-fold decrease in kcat/Km seen upon mutation. Indeed, in the related B1GH101 enzyme, a similar substrate model also suggests that the residue is involved in hydrogen bonding with the substrate (14).
The sugar binding specificity of the putative binding domain(s) was probed using two separate catalytically compromised mutants D764A/F. The mutations were made both to avoid cleavage of the glycans on the array and, in the case of the D764F mutant, to avoid active site binding. Only the disaccharide that functions as the substrate showed significant binding. This could reflect an adaptation to the galacto-N-biose rich mucin-covered environment that Streptococcus grows in. The binding of SpGH101 D764A/F to 3-O-sulphated disaccharide is interesting as this is a possible modification found in human mucin (32), although it is not known if this would be a substrate for SpGH101. The binding of the mutant E764F was weaker to the 3-O-sulphated disaccharide however both mutants showed large variations (100% standard deviation) in binding to this glycan, so it may not reflect a true ligand. Other weak binding to more complex ligands is seen in both experiments, but the importance of these interactions is unknown, and at this point it seems prudent not to over interpret such qualitative binding experiments. Low level binding to several glycans is common in these experiments, and more detailed binding studies using quantitative methods such as SPR or ITC will be required to evaluate the binding of individual glycans to the protein.
The strength of binding of the enzyme to the array suggests the binding is stronger than what would be seen only with an interaction through the active site, since the enzyme must stay bound during washing steps; this binding is more typical of a carbohydrate binding domain. Further analysis is required to determine where in the protein structure this binding is taking place, however an obvious target is the C-terminal CBM32 module. The CBM32 family has been shown to bind to various galactosides (see http://www.cazy.org/fam/CBM32.html) and this type of CBM is present in other cell surface enzymes from Streptococcus, although the specificity of the CBM32 modules is not yet known in these enzymes.
These GH101 proteins appear to be cell surface anchored which provides the opportunity to use carbohydrate binding as a tether for binding to host surfaces. It is clear that these enzymes have different substrate specificities (33) and more work on the various family members will be required to determine if the CBM like domains have the same specificity. Three dimensional structures of other related protein will be required to decipher how binding specificity is achieved by the various GH101 enzymes.
We acknowledge Dr. Hongming Chen for synthesis of substrates, Cynthia Bainbridge for technical assistance with the purification of proteins, and Dr. Chris Whitfield for his generosity during the preparation of the enzymes for the glycan array work. We thank Dr. David F. Smith at Core H of the Consortium for Functional Glycomics (National Institutes of Health Grant GM62116) for glycan array screening.
This work was funded by the Natural Sciences and Engineering Research Council of Canada and The Canadian Institutes for Health Research. R.Z thanks the BC Innovation Council for receipt of a scholarship and SGW thanks the Canada Research Chairs Program for salary support.