Compounds and reagents

Manno-oligosaccharides (mannobiose [M2], mannotriose [M3], mannotetraose [M4], mannopentaose [M5], and mannohexaose [M6]) and cello-oligosaccharides (cellotriose [G3], cellotetraose [G4], cellopentaose [G5], and cellohexaose [G6]) were purchased from Megazyme (Bray, Ireland). Cellobiose (G2) was purchased from Sigma-Aldrich (St. Louis, MO). Protein molecular weight markers for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad (Hercules, CA). High-performance liquid chromatography (HPLC) grade sodium acetate trihydrate was purchased from EMD Chemicals (Philadelphia, PA). All other reagents were purchased from Fisher Scientific (Hanover Park, IL) and were of the highest purity available.

Cloning and site-directed mutagenesis

Amplification of the man5B ORF (GenBank Accession ID: HM241690; protein ID: ADK22147) from C. polysaccharolyticus genomic DNA and cloning into the pET-46b Ek/LIC vector (Novagen, San Diego, CA) were described previously [6]. Amino acid substitutions (Y12A, Y12F, Y12Q, H84A, H84E, H84M, H84Q, N92A, N136A, R196A, and R196H) were constructed by introducing site-specific mutations into the coding sequence of man5B using either the QuikChange II or QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA) according to the supplier’s instructions. The pET46-Man5B plasmid [6] was used as the template, and mutagenic primers (Table S1 in File S3) were designed with the online QuikChange Primer Design program (https://www.genomics.agilent.com).

The PCR product (12.5 μL) was incubated with methylation-dependent restriction enzyme DpnI at 37 °C for 8 hours to digest the parental plasmid DNA. Escherichia coli XL10 competent cells were transformed with the product from the DpnI restriction digestion and plated onto lysogeny broth (LB) solidified with Bacto-agar (Difco) containing 100 μg mL^-1 ampicillin sodium salt, and the plates were incubated at 37 °C overnight. Individual colonies were cultivated in LB medium supplemented with ampicillin until stationary phase was reached, and plasmids were extracted using a QIAprep^® Spin Miniprep Kit (Qiagen, Valencia, CA). The presence of the site-directed mutations was confirmed by DNA sequencing (W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign).

Caldicellulosiruptor bescii DSM6725 ORF0234 (Athe_0234; GenPept Accession ID: YP_002572157) was amplified with PrimeStar^® DNA polymerase (Takara Bio Inc., Shiga, Japan) using primers Cb234F and Cb234R (Table S1 in File S3). The PCR product (cb234) was extracted from a 1% agarose gel using a QIAquick^® Gel Extraction Kit (Qiagen, Valencia, CA). The purified PCR product was treated with T4-DNA polymerase and dATP, annealed to the pET46b Ek/LIC vector (Novagen, San Diego, CA) according to the manufacturer’s instructions, and transformed into E. coli strain XL10 (Stratagene, La Jolla, CA). The transformed cells were plated on LB agar plates supplemented with ampicillin. Plasmids were extracted from the transformed cells, and the integrity of cb234 was confirmed by DNA sequencing as described above.

DNA oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). E. coli BL21-CodonPlus(DE3)-RIPL cells (Agilent Technologies, Inc., Santa Clara, CA) were used for protein production. E. coli strains XL10 and DH5α were used to propagate the recombinant plasmids.

Recombinant protein production and purification

E. coli BL21-CodonPlus(DE3)-RIPL cells harboring a recombinant plasmid bearing either the gene for wild-type CpMan5B, mutants of CpMan5B, or CbMan5D were cultured in 10-mL LB containing 100 μg mL^-1 sodium-ampicillin and 50 μg mL^-1 chloramphenicol for approximately 8 hours at 37 °C with vigorous shaking. The initial 10-mL cultures were used to inoculate LB (1 L) containing the two antibiotics, at the same concentrations stated above, in a 2.8-L Fernbach flask. The cultures were incubated at 37 °C at 200 rpm until the absorbance at 600 nm reached ≈0.3 (after approximately 2.5 hours). Gene expression in E. coli cultures was then induced with the addition of IPTG (0.1 mM, final). The temperature was decreased to 16 °C, and the cultures were allowed to incubate for an additional 16 hours at 200 rpm. Cell pellets were harvested from the cultures by centrifugation at 4,651 × g for 30 minutes at 4 °C, washed with 35 mL binding buffer (50 mM Tris, 300 mM NaCl, pH 7.5), and frozen as cell pellets at -80 °C.

Frozen cell pellets were thawed and re-suspended in 35-mL ice-cold binding buffer and passed through an EmulsiFlex C-3 homogenizer (Avestin, Ottawa, Canada) to rupture the cells. The cell lysates were clarified by centrifugation at 12,857 × g for 20 min at 4 °C, and the supernatant containing the soluble fraction was recovered.

For purification of wild-type or mutant CpMan5B and CbMan5D, the cell-free extracts were incubated at 65 °C for 30 min and centrifuged at 12,857 × g for 20 min at 4 °C to pellet the precipitated heat-labile E. coli proteins. Cloning of the genes fused each to a hexa-histidine tag encoded in the plasmid. Thus, each protein was purified from the resulting supernatants using Talon metal affinity resin (Clontech, Mountain View, CA) according to the manufacturer’s instructions. Briefly, the resin was incubated with the lysate for one hour to allow binding of the recombinant protein. The resin was then washed with ten column volumes of binding buffer, and the protein was eluted with elution buffer (50 mM Tris, 300 mM NaCl, 250 mM imidazole pH 7.5). The buffer of the eluate containing the recombinant protein was exchanged by concentrating the protein in Spin-X UF 20-mL centrifugal concentrators with molecular mass cutoffs of 10 kDa (Corning, Lowell, MA). The protein in the eluate buffer was twice diluted >20-fold into protein storage buffer (50 mM Tris, 150 mM NaCl, pH 7.5).

Protein concentrations were determined using a spectrophotometer according to a standard method [8]. Briefly, the absorbance at 280 nm (A₂₈₀) of the recombinant protein solutions was determined using a NanoDrop 1000 from Thermo Fisher Scientific Inc. (Waltham, MA). The molecular masses of the recombinant proteins were predicted using the online ProtParam tool (http://web.expasy.org/protparam/). The values of A₂₈₀, molecular mass, and extinction coefficient (Table S2 in File S3) were used to calculate the concentration of each purified protein.

Crystallization, data collection and model refinement

Initial screening of crystallization conditions for CpMan5B was performed by the sitting-drop vapor diffusion method using a nanodrop dispenser, Mosquito (TTP Labtech), and two crystallization screening kits, Crystal Screen and INDEX (Hampton Research). Each crystallization drop was made by mixing 200 nL of the concentrated protein solution and an equal volume of reservoir solution. Plate-like crystals were obtained from a drop with a reservoir containing 0.2M ammonium phosphate monobasic, 0.1 M Tris (pH 8.5) and 50% (v/v) (+/-)-2-methyl-2,4-pentanediol (MPD) (Crystal Screen H7). Diffraction quality crystals were reproducibly obtained by the hanging-drop vapor diffusion method in a larger scale manual setup, where 1 μL of the protein solution was mixed with an equal volume of the reservoir solution and equilibrated against 500 μL of the reservoir at 20 °C. Better crystals grew to a maximum dimension of 100 μm in two weeks.

X-ray Diffraction data were collected at BL38B1 in SPring-8 (Harima, Japan). The CpMan5B crystals were picked up from the crystallization drops and then flash-cooled in a liquid nitrogen gas stream. No cryoprotection was required because the crystallization drops contained a high concentration of MPD. The collected data were processed using the HKL2000 package [9].

The CpMan5B structure was determined by the molecular replacement method with the program CNS [10], using the atomic coordinates of Thermotoga maritima endo-glucanase (PDB code 1VJZ), of which sequence identity is 41%, as a probe. The crystals belong to the monoclinic space group P2₁, with the unit cell dimensions of a = 50.133 Å, b = 147.642 Å, c = 55.422 Å, and β = 104.51°, and contained two CpMan5B molecules in the asymmetric unit. The two correctly positioned molecules were refined with CNS and O [11] until convergence. The crystallographic data and refinement statistics are summarized in Table 1. The three dimensional coordinates are deposited in the Protein Data Bank under code 3W0K.

Biochemical assay of wild-type and mutant CpMan5B and CbMan5D

Enzymatic activities of wild-type CpMan5B and its derivative mutant proteins were determined at 65 °C in 50 mM sodium citrate buffer containing 150 mM NaCl with pH 5.5 in accordance with the previously reported temperature and pH optima for the wild-type enzyme [6] using M6 and G6 as substrates. The enzymatic activity of CbMan5D was assayed in the same buffer as wild-type CpMan5B but at 75 °C. For assays to be analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), the enzymes were added to the mixtures at a final concentration of 0.5 μM and incubated for 8 hours in a total volume of 50 μL with G6 (5 mg mL^-1) as substrate or at a final concentration of 0.1 μM and incubated for 10 minutes in a total volume of 30 μL with M6 (5 mg mL^-1) as substrate. The reactions were heat-inactivated for 10 minutes at 99.9 °C, diluted into distilled, deionized water (9-fold for G6 reactions; 16-fold for M6 reactions), and centrifuged at 15,871 × g for 5 minutes. The lengths of reaction time were selected from preliminary experiments performed with wild-type CpMan5B enzyme that identified a period in which production of the mono-, di-, and tri-saccharides was linear over time (data not shown).

The end products were analyzed by HPAEC-PAD using a System Gold^® HPLC instrument from Beckman Coulter (Fullerton, CA) equipped with a CarboPac PA1 guard column (4 × 50 mm), a CarboPac PA1 analytical column (4 × 250 mm) from Dionex Corporation (Sunnyvale, CA), and a Coulochem^® III electrochemical detector from ESA Biosciences (Chelmsford, MA). The elution condition for cello-oligosaccharides was a linear gradient from 0-150 mM sodium acetate in 100 mM NaOH over 15 min. The elution condition for manno-oligosaccharides was a linear gradient from 0-50 mM sodium acetate in 100 mM NaOH over 30 min [12,13]. Eluted saccharide products were identified and quantified by comparing peak retention times and peak areas from the chromatographs of commercially available mono- and oligo-saccharides as standards.

Overall structure of CpMan5B

The structure of CpMan5B was determined by the molecular replacement method and refined at 1.6 Å resolution (Figure 1). The protein is well-ordered in the crystal, and, therefore, almost all amino acid residues were visible in the electron density map, except for a short segment encompassing residues 212-216 of chain B. There are two CpMan5B molecules in the asymmetric unit, and the two molecules exhibit almost the same structure, with a relative RMSD of 0.21 Å for the corresponding 325 Cα atoms. CpMan5B folds into an (α/β)₈ barrel with a deep active site cleft on the carboxy-terminal side of the central β-sheet, which is typical for many carbohydrate hydrolyzing proteins (Figure 1A). Figure 1B shows the molecular surfaces of CpMan5B colored according to the electrostatic potential. The substrate-binding cleft is widely open at both the non-reducing and reducing ends toward solvent, which is a typical feature for endo-acting carbohydrate degrading enzymes.

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Figure 1. Crystal structure of C.

polysaccharolyticus Man5B.

A, The structure is shown as a ribbon representation with a color spectrum from blue (N-terminus) to red (C-terminus). B, Orthogonal views of the molecular surfaces of CpMan5B colored according to the electrostatic potential. C, Close-up view of the Tris molecule bound to the active site of CpMan5B. The Tris molecule and the contacting residues are highlighted by a stick representation.

https://doi.org/10.1371/journal.pone.0080448.g001

Incidentally, a Tris molecule, which was supplied from the crystallization reservoir solution, is tightly bound to the active site, and forms hydrogen bonds with five amino acid residues, H84, N92, N136, E137 and E258 (Figure 1C). Residues E137 and E258 were previously determined to be the catalytic residues by sequence alignment and enzymatic assays of site-directed mutants [6], and roles in catalysis for residues equivalent to H84 and N136 have been described for other enzymes [14-17]. In particular, N92 is positioned on the β4-α4 loop, which is usually disordered in the related enzyme structures because of potential flexibility. In the CpMan5B structure, however, this loop is well ordered, possibly due to the hydrogen bond between N92 and the bound Tris molecule.

The structure of CpMan5B is similar to GH5 mannanases and endo-glucanases with known structures. A search of the Dali Database [18] revealed that CpMan5B exhibits highest structural similarity to the mannan-inducible [19] Thermotoga maritima glucomannanase [20] (Tm1752) (1VJZ; 41% amino acid identity, RMSD 1.7 Å for corresponding 325 Cα atoms), although, to our knowledge, mannanase activity of Tm1752 has not been demonstrated in vitro. CpMan5B exhibits lower similarity to a Clostridium thermocellum cellulase (CtCel5C) (1CEN; 28%, 2.2 Å for 334 Cα atoms) [21], the dual specificity mannanase/endo-glucanase from T. maritima (Tm1751; TmCel5A) (3AMD; 23%, 2.3 Å for 312 Cα atoms) [22], and an endo-glucanase from the thermophilic bacterium, Fervidobacterium nodosum (3RJY; 22%, 2.4 Å for 308 Cα atoms) [23].

Amino acid determinants for binding gluco-configured substrates

Superposition of the Clostridium thermocellum Cel5C-cellobiose structure (PDB 1CEN) [21] with CpMan5B (Figure 2) revealed overall conservation in protein fold (Figure 2A). In the CtCel5C-cellobiose complex, two β-1,4-linked glucose molecules occupy the -2 and -1 subsites (according to the nomenclature by Davies et al. [24]) in the active site, and most of the residues contacting the bound sugars are conserved in CpMan5B, possibly providing an explanation for the endo-glucanase activity observed for CpMan5B.

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Figure 2. Structural comparison of CpMan5B with Clostridium thermocellum cellulase (CtCel5C).

A, The CtCel5C-cellobiose complex (green) is superimposed onto CpMan5B (tan). B, Close-up view of the active site. The figure is drawn in the same orientation as in A. The cellobiose molecule bound to CtCel5C is shown in a stick model colored yellow and red for carbon and oxygen atoms, respectively. Contacting residues of CtCel5C (green), and the corresponding residues of CpMan5B (tan) are shown in panel B. Sugar subsites are indicated in black font.

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At subsite -1 of CtCel5C, the enzyme makes a number of hydrogen bond contacts to the glucose moiety. These contacts include E140 and E280 with the C1 and C2, hydroxyls, respectively, and bidentate hydrogen bonds to the C3 hydroxyl mediated by Q16 and H90 and to the C6 hydroxyl mediated by W212 and H207. These residues are present in CpMan5B as E137, E258, Y12, H84, W210, and H205, respectively (Figure 2B). With the exception of Q16/Y12, these residues are conserved in GH5 enzymes and are unlikely to significantly alter substrate specificity. At subsite -2, the W313 sidechain Nε and M318 mainchain O atoms form hydrogen bonds with the C2 hydroxyl. It is possible that CtCel5C W313 could also hydrogen bond with an axial OH group at C2 of a mannose moiety, if mannose were bound to the subsite -2 instead of glucose at a similar location and with a similar conformation. Three other hydrogen bond contacts in the -2 subsite include D319 with the C3 hydroxyl and Q16 with the ring oxygen and the C6 hydroxyl of the glucose moiety. Amino acid residue W313 is conserved in CpMan5B as W291, but D319 and Q16 are replaced with P296 and Y12 in CpMan5B, respectively. In addition, two tryptophan residues (W313 and W212) in CtCel5C are co-planar to the sugar residues in the -1 and -2 subsites and thereby help to stabilize these sugars by hydrophobic stacking interactions. These residues are conserved in CpMan5B as W291 and W210. Thus, most of the key residues that stabilize the cellobiose molecule in the CtCel5C-cellobiose structure are conserved in CpMan5B.

Despite the similarities, several key differences at the -1 and -2 binding subsites could help to explain the differences in specificity between CtCel5C and CpMan5B. The β8-α8 loop, on which CtCel5C M318 and D319 are located, is shorter in CpMan5B. Therefore, the hydrogen bonds between the β8-α8 loop and the sugar occupying subsite -2 in CtCel5C would be absent in CpMan5B, unless a large conformational change of the loop occurs upon substrate binding. Although Q16 of CtCel5C is replaced by Y12 in CpMan5B, it is located in a similar spatial position so that the hydroxyl group of the tyrosine residue could hydrogen bond with the C3 hydroxyl of the -1 subsite-bound sugar, but not with the ring oxygen of the -2 subsite-bound sugar.

Another difference between the two active sites is the presence of N92 in CpMan5B which is contained on the loop between β-strand 3 and α-helix 3. The side chain of N92 faces towards the active site and is positioned to make hydrogen bond contacts with the C6 hydroxyl of the glucose moiety at the -2 subsite. Amino acid sequence alignments reveal Q98 as a corresponding residue in CtCel5C, but in the CtCel5C-cellobiose structure, this residue showed poor electron density definition [21], thereby preventing a direct comparison. The large and diverse family of GH5 enzymes was recently categorized into 51 distinct subfamilies [25], and CtCel5C belongs to subfamily GH5_37 while CpMan5B belongs to subfamily GH5_36. The CtCel5C Q98 residue is not conserved in the GH5_37 subfamily (only 1 of 11 unique sequences with identity <99%), but interestingly, the 27 enzymes in the subfamily GH5_36 show strong conservation (89%) of an asparagine at position 92 (CpMan5B numbering, data not shown), suggesting that this residue may fulfill a unique role in this subfamily. Another difference between the active sites of CtCel5C and CpMan5B is the replacement of H198 with R196. In CtCel5C, H198 forms a 2.7 Å hydrogen bond with the catalytic acid-base catalyst, E140, which is mutated to a glutamine in the CtCel5C structure. In the CpMan5B structure, R196 adopts a conformation that permits a hydrogen bond to form between its Nε atom and the Oε₂ of the catalytic glutamate, E137. Interestingly, this hydrogen bond is also 2.7 Å in length, suggesting that R196 is sufficient to carry out the same function as H198. As discussed later, this observation is of considerable significance as a histidine at this position has been implicated as being critical for catalysis for GH5 enzymes [23].

Amino acid determinants for binding manno-configured substrates

The superposition of the endo-glucanase CtCel5C and the β-mannanase CpMan5B, which has a minor endo-glucanase activity, suggested that there are several differences in active site residues between the two enzymes. To evaluate whether these residues may correspond to an increased β-mannanase activity, CpMan5B was aligned with the TmCel5A-mannotriose structure [22]. In the TmCel5A-mannotriose complex, three mannose sugars occupy the -3, -2, and -1 subsites in the active site, hereto referred to as Man^(-1), Man^(-2) and Man^(-3) (Figure 3).

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Figure 3. Structural comparison of CpMan5B with Thermotoga maritima cellulase (TmCel5A).

A, The TmCel5A-mannotriose complex (green, PDB ID: 3AZS) is superimposed onto CpMan5B (tan). B, Close-up view of the active site. The mannotriose molecule bound to TmCel5A is shown by a stick model colored yellow and red for carbon and oxygen atoms, respectively. Contacting residues of TmCel5A (green), and the corresponding residues of CpMan5B (tan) are shown in panel B. Sugar subsites are indicated in black font. C. The active site has been rotated 180° around the y-axis relative to the view in panel B.

https://doi.org/10.1371/journal.pone.0080448.g003

In the -1 and -2 subsites, many of the hydrogen bond contacts are conserved in comparison with the TmCel5A-mannotriose structure with several notable differences. These include the replacement of H96 in TmCel5A with R85 in CpMan5B, which appears to point away from the active site, thereby eliminating a hydrogen bond to the Man^(-1) C3 hydroxyl. Interestingly, this hydrogen bond contact is replaced by one with Y12 in CpMan5B. Y12 corresponds to E23 in TmCel5A, but the latter did not appear to contact the mannose ligands. Furthermore, the Y12 residue binds to the Man^(-2) C2 hydroxyl and hemiacetal oxygen. Notably, N20 in TmCel5A, which forms a hydrogen bond contact with the Man^(-2) C2 hydroxyl and has been suggested to be an important determinant of mannanase activity [7], is replaced with Q9 in CpMan5B. The β-carbon of Q9 in CpMan5B is closely approximated (2.0Å) to the α-carbon of N20 in TmCel5A (Figure 3C). It is likely, therefore, that when mannose is bound in the -2 subsite, Q9 forms a hydrogen bond with the C2 hydroxyl. Therefore, in the CpMan5B structure, there is one additional hydrogen bond contact to the Man^(-2) sugar mediated by Y12. This residue could in part explain the increased selectivity of CpMan5B for mannans over glucans as compared to TmCel5A.

Mutational analyses of active site residues

The crystal structure of CpMan5B and comparisons to the three dimensional structures of CtCel5C (subfamily GH5_37) and TmCel5A (subfamily GH5_25) revealed important differences in active site residues. Several residues (Y12, H84, N92, N136, and R196) were changed by site-specific mutagenesis, resulting in thirteen mutant proteins (Figures S1-S2 in File S1, Table S3 in File S3) with relative molecular masses of ≈41kDa, so that their roles in enzyme function could be evaluated.

Amino acid substitutions in residues H84, N92, and N136, which form hydrogen bonds with the Tris molecule in the CpMan5B crystal structure, had dramatic effects on enzyme activity for both substrates. Four amino acid substitutions (A, E, M, and Q) at residue H84 and an alanine substitution at residue N136 in CpMan5B virtually eliminated all activity with M6 and G6 (Figure 4) indicating that these two residues are essential to both the mannanase and cellulase activities. As mentioned above, H84 and N136 have been implicated previously in the activities of other β-1,4-endoglucanases. The activities of mutant enzyme CpMan5B^N92A were also reduced, although some activity with both substrates was detected.

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Figure 4. Specific activities of wild-type and mutant CpMan5B enzymes and the CbMan5D enzyme.

Rates of product formation from either mannohexaose (Panels A, C) or cellohexaose (Panels B, D) were determined after 10 minutes and 8 hours, respectively, when the rates of product formation by the wild-type enzymes were linear with time. The amino acid changes in the x-axes labels indicate the site-specific mutants of the CpMan5B enzyme. Parenthetical values above the bars show the percentage of wild-type CpMan5B activity, and asterisks indicate that the raw data are significantly different (P<0.05, Student's paired t test) from those of the wild-type CpMan5B enzyme in the same experiment. Abbreviations: M1, mannose; M2, mannobiose; M3, mannotriose; G1, glucose; G2, cellobiose; G3, cellotriose; n.d., end product(s) was not detected under these assay conditions; IU, international units (μmol min^-1 mg^-1); mIU, milli-international units (nmol min^-1 mg^-1). Lower limits for detection were <2 IU mg^-1 for mannosaccharides and <2 mIU mg^-1 for cellosaccharides.

https://doi.org/10.1371/journal.pone.0080448.g004

The residue at position 196 is highly conserved in GH5 enzymes as a histidine [26] and is thought to participate in an electron relay network that is critical for catalysis [7]. Surprisingly, the corresponding residue in CpMan5B is an arginine, and this residue is, in fact, 100% conserved in subfamily GH5_36 (data not shown). Therefore, to evaluate the role of this residue in catalysis, R196A and R196H mutations were made. The R196A mutation eliminated all detectable activity with both substrates, indicating that this residue is essential for catalysis. The CpMan5B^R196H enzyme, however, retained some activity with both G6 and M6 substrates (7% and 14% of CpMan5B^WT, respectively) suggesting that the histidine and arginine are interchangeable at this position.

The structure of CpMan5B and its comparison with the structures of other GH5 enzymes suggested that Y12 could be important for imparting increased β-mannanase activity to CpMan5B. An amino acid sequence alignment of the 27 subfamily GH5_36 enzymes, including CpMan5B, from the GenBank database revealed that this position was replaced with a phenylalanine in 20 enzymes (data not shown). We, therefore, decided to test the effect of a phenylalanine at this position. In addition, less conservative changes, Y12A and Y12Q, were also tested. The Y12A, Y12F, and Y12Q mutations each resulted in moderate losses of activity on both G6 and M6 substrates (Figure 4, Table S4 in File S3). However, CpMan5B^Y12A retained 28% of the WT mannanase activity and 67% cellulase activity, but the trend was reversed for the CpMan5B^Y12F mutant enzyme, which retained 63% mannanase and 29% cellulase activities. The CpMan5B^Y12Q mutant enzyme lost activities proportionately (retaining only ≈20%) for both G6 and M6 substrates. Taken together, these data indicate that the residue at position 12 can alter the selectivity for glucose- or mannose-configured substrates with CpMan5B^Y12F retaining the highest preference for mannose-configured substrates. Incidentally, double mutants of the Y12F and Y12Q mutations with the R196H mutation (CpMan5B^Y12F/R196H and CpMan5B^Y12Q/R196H) were constructed, but the activities of these double mutants were very low or undetectable. Therefore, their combined effects on substrate specificity could not be evaluated.

Identification of C. bescii Man5D as a GH5 enzyme with mannanase and cellulase activities

An alignment of subfamily GH5_36 enzymes revealed that Y12 is conserved in only 5 of 27 enzymes and is replaced frequently by a phenylalanine. Biochemical analysis showed that the Y12F mutant had improved selectivity for manno-oligosaccharides, and we therefore hypothesized that F12 could be used as a probe for enzymes with relatively high mannanase activity. A BLASTp search of the GenBank database revealed ORF0234 (CbMan5D: Genbank Accession ID: ACM59384) from C. bescii as a hyperthermophilic putative cellulase with 40.1% identity to CpMan5B. CbMan5D, also in subfamily GH5_36, contains a phenylalanine at the site corresponding to residue Y12 of CpMan5B, but contained identical residues at positions corresponding to H84, N92, N136, and R196. Preliminary analyses of recombinant CbMan5D showed that this enzyme had optimal pH and temperature of 5-5.5 and 75-80°C, respectively (data not shown). CbMan5D was also assayed qualitatively using thin-layer chromatography (TLC, Figure S3 in File S2). CbMan5D hydrolyzed mannose-configured substrates with a degree of polymerization (DP) ≥ 2, which was similar to CpMan5B. CbMan5D also degraded glucose-configured substrates with a DP ≥ 4, differing only slightly from CpMan5B, which could also degrade G3 [6]. Thus, CbMan5D appeared to be a mannanase with some endoglucanase activity similar to CpMan5B. Interestingly, a sequence alignment shows that CbMan5D has an arginine (position 139 according to numbering in Figure 5) which is semi-conserved in the GH5 family and has been shown to bind the +2 mannosyl specifically and to be functionally important [27-29]. The corresponding arginine is not present in CpMan5B (Figure 5).

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Figure 5. Amino acid sequence alignment of CpMan5B, CbMan5D, and TmCel5A.

The three polypeptides were aligned using ClustalW with the Blosum62 similarity matrix (http://www.genome.jp/tools/clustalw/). Boxes indicate similar residues and dark shading indicates identical residues. The GenBank accession numbers for CpMan5B, CbMan5D, and TmCel5A are ADK22147, ACM59384, and AAD36816, respectively.

https://doi.org/10.1371/journal.pone.0080448.g005

To compare the relative activities of CbMan5D to those of CpMan5B, CbMan5D was assayed quantitatively using a HPAEC-PAD method similar to that used for CpMan5B, but carried out at 75 °C (Figure 4A-B). The data showed that CbMan5D is also primarily a β-mannanase with a capacity to cleave β-1,4-glucose linkages. To assess substrate specificity, the ratios of β-mannanase:endo-glucanase specific activities for CpMan5B^WT, CpMan5B^Y12F and CbMan5D were calculated as 310, 669, and 420, respectively. The higher ratio values of CpMan5B^Y12F and CbMan5D relative to CpMan5B^WT suggest that the residue at this position may, in part, influence the relative mannanase:endoglucanase activities in subfamily GH5_36 enzymes.