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A Novel Glycoside Hydrolase Family 113 Endo-␤-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family Wei Xia,a,b Haiqiang Lu,c Mengjuan Xia,a Ying Cui,a Yingguo Bai,a Lichun Qian,b Pengjun Shi,a Huiying Luo,a Bin Yaoa Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of Chinaa; College of Animal Science, Zhejiang University, Hangzhou, People’s Republic of Chinab; College of Food Science and Technology, Hebei Agricultural University, Baoding, People’s Republic of Chinac

Few members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-␤-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilic Alicyclobacillus sp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counterpart Alicyclobacillus acidocaldarius ManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ⬇ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of ␤-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support ⴚ4 to ⴚ1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 ␤-mannanases and distinguishes them from counterparts of other families.

A

major component of hemicellulose in the plant cell walls of softwood, plant seeds, and beans is ␤-1,4-mannan, including mannan polysaccharides, glucomannan, galactomannan, and galactoglucomannan. It is composed of a backbone of ␤-1,4-linked mannose or a combination of glucose and mannose with side chains of ␣-1,6-linked galactose residues (1). The mannan-degrading enzymes have biotechnological applications in various areas, such as feed manufacturing (2), paper processing (3), and coffee extract treatment (4). Mannooligosaccharides, the major hydrolysis products of mannan, are beneficial as animal nutrition additives due to their potential prebiotic properties (5). The complete degradation of mannan polysaccharides into monomers requires an enzyme system including ␤-mannanase (EC 3.2.1.78), ␤-mannosidase (EC 3.2.1.25), ␤-glucosidase (EC 3.2.1.21), and ␣-galactosidase (EC 3.2.1.22). Of these, ␤-mannanase randomly hydrolyzes the ␤-D-1,4-mannopyranoside linkages and plays the major role in mannan degradation. ␤-Mannanases are widely distributed in various organisms, including bacteria, yeasts, filamentous fungi, and plants. Based on the amino acid sequence and structural similarities of catalytic domains, ␤-mannanases are grouped into three glycoside hydrolase (GH) families in the Carbohydrate-Active enZYmes (CAZy) database (6), i.e., 5, 26, and 113. GH 113 is a newly defined family that belongs to GH clan A and shares the same (␤/␣)8 folding structure and catalytic machinery. Two glutamate residues at the C termini of ␤4 and ␤7 sheets serve as a general acid/base and a nucleophile to catalyze the cleavage of glycosidic bonds via a retaining double-displacement mechanism. As yet, 180 sequences have been classified into GH 113 (http://www.cazy.org/GH113 .html); moreover, only one member, AaManA from Alicyclobacil-

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lus acidocaldarius Tc-12-31, has been functionally characterized and its structure resolved (7). AaManA is an intracellular endo-␤1,4-mannanase with transglycosylation activity, and several residues related to substrate binding are identified by sequence alignments and structure analysis. However, the catalytic mechanism and enzymatic properties of GH 113 have not yet been clarified. In the present study, another GH 113 ␤-1,4-mannanase gene (man113A) was cloned from the thermoacidophilic bacterium Alicyclobacillus sp. strain A4 (8) and expressed in Escherichia coli. The catalytic properties and residues related to substrate recognition of Man113 were determined by biochemical characterization and site-directed mutagenesis. We provide here valuable information regarding the functions of GH 113 mannanases.

Received 23 December 2015 Accepted 20 February 2016 Accepted manuscript posted online 26 February 2016 Citation Xia W, Lu H, Xia M, Cui Y, Bai Y, Qian L, Shi P, Luo H, Yao B. 2016. A novel glycoside hydrolase family 113 endo-␤-1,4-mannanase from Alicyclobacillus sp. strain A4 and insight into the substrate recognition and catalytic mechanism of this family. Appl Environ Microbiol 82:2718 –2727. doi:10.1128/AEM.04071-15. Editor: R. M. Kelly, North Carolina State University Address correspondence to Bin Yao, [email protected], or Pengjun Shi, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.04071-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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MATERIALS AND METHODS Strains, media, vectors, and chemicals. The donor strain Alicyclobacillus sp. strain A4 (CGMCC3147) was obtained from the China General Microbiological Culture Collection Center. Escherichia coli Trans1-T1 and the vector pEASY-T3 from TransGen (China) were used for gene cloning and sequencing, respectively. E. coli BL21(DE3) (TransGen) and the vector pET-30a(⫹) (Novagen, USA) were used for heterologous protein expression. The DNA purification kit, restriction endonucleases, and LA Taq DNA polymerase were purchased from TaKaRa (Japan). The highfidelity DNA polymerase Fast Pfu was purchased from TransGen. T4 DNA ligase was purchased from Promega (USA). Locust bean gum, konjac flour, ivory nut mannan, guar gum, barley ␤-glucan, birchwood xylan, and carboxymethyl cellulose sodium salt (CMC-Na) were obtained from Sigma-Aldrich (USA). Mannooligosaccharide standards (mannose, mannobiose, mannotriose, mannotetrose, mannopentaose, and mannohexaose) and high-viscosity guar galactomannans containing 21, 28, 34, or 38% galactose were purchased from Megazyme (Ireland). All other chemicals were of analytical grade. Gene cloning and sequence analysis. The ␤-mannanase gene man113A was amplified from the genomic DNA of Alicyclobacillus sp. A4 by using the primers man113EF and man113ER (shown in Table S1 in the supplemental material), which were designed in accordance with the partial genome sequence (data unpublished; the GenBank accession number for this gene is KC460333.1). PCR products were purified and ligated into the pEASY-T3 vector and then transformed into E. coli Trans1-T1 for sequencing. Nucleotide and deduced amino acid sequences were aligned using the BLASTn and BLASTp programs (http://www.ncbi.nlm.nih.gov /BLAST/), respectively. Vector NTI Advance 11.5 software (Invitrogen) was used to analyze the nucleotide sequence, predict the molecular weight of the deduced protein, and align multiple sequences. The signal peptide was predicted by the SignalP 4.1 server (http://www.cbs.dtu.dk/services /SignalP/). The neighbor-joining phylogenetic tree based on the deduced amino acid sequences was built using the MEGA software (version 6.0). Expression and purification of recombinant Man113A. The coding sequence of man113A was amplified by PCR using the primers Man113EF and Man113ER (see Table S1 in the supplemental material), and the PCR products were purified as described above. Both the purified PCR products and pET-30a(⫹) were digested by EcoRI and HindIII and ligated downstream of the His tag coding sequence. The recombinant plasmids were then individually transformed into E. coli BL21(DE3)-competent cells. The positive transformants were screened and checked by DNA sequencing. Man113 expression in E. coli BL21(DE3)-competent cells was induced at 30°C for 6 h by 0.6 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) at the end of logarithmic growth. Cultures were centrifuged at 12,000 ⫻ g and 4°C for 5 min. The cells (⬃5 g) were resuspended in 25 ml of lysis buffer (20 mM Tris-HCl [pH 7.0]) and disrupted by sonication on ice with 100 cycles of 5-s short bursts at 200 W and 3 s of cooling by use of an ultrasonic cell disruptor (Scientz, China). The cell debris was removed by centrifugation, and the supernatant was subjected to Ni2⫹-NTA chromatography with a linear gradient of imidazole (2 to 300 mM) in 50 mM Tris-HCl– 0.5 M NaCl (pH 7.6). Fractions exhibiting ␤-mannanase activity were pooled and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Elution peaks of a single band with the predicted size were recovered and concentrated as a purified enzyme solution. The protein concentration was determined by a Bradford assay with bovine serine albumin as a standard. Assay of the enzymatic activity. ␤-Mannanase activity was assayed by measuring the amount of reducing sugars released from locust bean gum using the 3,5-dinitrosalicylic acid (DNS) method (9). The standard reaction system contained 100 ␮l of appropriately diluted enzyme sample and 900 ␮l of 0.5% (wt/vol) locust bean gum in 100 mM Na2HPO4-citric acid (pH 6.0). The reactions were carried out at 50°C for 10 min and terminated by the addition of 1.5 ml of DNS reagent. After 5 min of boiling in water, the mixtures were cooled to room temperature and the absorbance at 540 nm was measured. For the control sample, the recombinant en-

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zyme was added after DNS reagent. One unit of ␤-mannanase activity was defined as the amount of enzyme that released 1 ␮mol of reducing sugar per min under standard assay conditions. Each experiment was performed in triplicate. Biochemical characterization of purified recombinant Man113A. The optimal pH for Man113A activity was determined at 50°C for 10 min over a pH range of 2.0 to 11.0 using the following buffers: 100 mM Na2HPO4-citric acid (pH 2.0 to 8.0), 100 mM Tris-HCl (pH 8.0 to 9.0), and 100 mM glycine-NaOH (pH 9.0 to 11.0). To estimate the pH stability, the enzyme was appropriately diluted in the buffers mentioned above and preincubated at 37°C for 1 h without substrate, and the residual activities were measured under the standard conditions (pH 6.0 and 50°C for 10 min). The optimal temperature of Man113A was examined at the optimal pH by measuring the enzyme activity over the temperature range of 20 to 90°C. The thermostability was investigated by determining the residual enzyme activity after preincubation at 40, 50, and 60°C and the optimal pH without substrate for various time periods. The samples were collected at 0, 2, 5, 10, 20, 30, and 60 min, respectively. Enzyme activity assays were performed under the standard conditions. The enzyme activity of Man113A was measured in the presence of 1 or 5 mM (final) concentrations of various metal ions and chemical reagents [NaCl, KCl, CaCl2, LiCl, CoCl2, CrCl3, NiSO4, CuSO4, MgSO4, FeSO4, MnSO4, ZnSO4, Pb(CH3COO)2, AgNO3, HgCl2, EDTA, SDS, and ␤-mercaptoethanol] to estimate their effects on Man113A activity. The reaction without any additive was used as a blank control. Each experiment was performed in triplicate. Substrate specificity. To investigate the substrate specificity of Man113A, activity assays were performed under optimum conditions with 0.5% (wt/vol) locust bean gum (galactomannan, 4:1), konjac flour (glucomannan), ivory nut mannan, guar gum, barley ␤-glucan, birchwood xylan, CMC-Na, and guar galactomannan containing 21, 28, 34, and 38% galactose as the substrate. Calculation of the specific activity of each substrate used the standard curve corresponding to each monosaccharide component. Hydrolysis of mannan and mannooligosaccharides. Products of mannan and mannooligosaccharide hydrolysis by Man113A were analyzed using high-performance anion-exchange chromatography (Thermo Fisher Scientific, USA) with a Carbo-Pac PA200 column (3 ␮m by 250 mm). For mannans, purified recombinant Man113A (3 U) was incubated in a 1-ml reaction system containing 1% (wt/vol) locust beam gum or konjac flour at 60°C for various durations. For mannooligosaccharides, the experiments were carried out with 1 U/ml enzyme (0.085 ␮M) and a substrate concentration of 30 ␮M. Samples (500 ␮l) were withdrawn at regular intervals, heated in a boiling water bath for 5 min to terminate the reaction, and centrifuged at 12,000 ⫻ g and 4°C for 10 min, followed by ultrafiltration using a Vivaflow 200 membrane with a 5-kDa molecular mass cutoff (Vivascience, USA) to remove the macromolecules. The hydrolysates were diluted 100-fold in double-distilled H2O (ddH2O), and 25 ␮l of each sample was injected into the column. NaOH (100 mM) was used to elute the oligosaccharides at a flow rate of 0.3 ml/min. Calibration curves were plotted using ␤-1,4-mannooligosaccharides as standards. The amount of each mannooligosaccharide was calculated based on the peak area of each standard sample. The catalytic efficiencies of Man113A and its mutants against mannooligosaccharides were calculated by fitting the data to the following equation: catalytic efficiency (kcat/Km) ⫽ ln([S0]/[St])/E/time, where [S0] represents the initial substrate concentration, [St] represents the substrate concentration at a specified time during the reaction, and [E] represents the enzyme concentration (10, 11). Transglycosylation activity of Man113A. The thin-layer chromatography (TLC) method was used to investigate the transglycosylation activity of Man113A with mannooligosaccharides (mannotriose, mannotetraose, mannopentaose, and mannohexaose) as both donors and acceptors. Each reaction system containing 50 mM individual mannooligosaccharide and 1 U of Man113A/ml was incubated at pH 6.0 and 37°C

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for 4 h. After the removal of Man113 by ultrafiltration, the reaction mixtures were diluted 50⫻ in ddH2O. The mannooligosaccharide standards and 3 ␮l of each diluted sample were spotted on an aluminum-coated silica gel 60 sheet (Merck, Germany), developed in a system of 2:1:1 (vol/ vol/vol) n-butyl alcohol/acetic acid/water, colored by saturating the plate in 5% sulfuric acid (vol/vol), and then heated at 110°C for 5 min. Homology modeling and molecular docking of Man113A. The tertiary structure of the deduced Man113A was homology modeled using the Discovery Studio v2.5 software (Accelrys, USA) with the crystal structure of AaManA from A. acidocaldarius (PDB code 3CIV, 55% identity) as the template and refined by the embedded energy minimization program and Verify-3D. The molecular docking of modeled Man113A and mannohexaose was carried out by AutoDock Vina (12) to elucidate the substrate binding interaction. The docking grid, with an appropriate size of 40 Å by 40 Å by 40 Å, was set to center the ␤-carbon atom of Glu144, which was predicted to be the nucleophile of Man113A. The docked complexes were analyzed based on previous studies (7, 13) to select the most reliable binding conformation. Three-dimensional molecular visualization and figure preparation were performed with PyMOL (version 1.7.2.1; Delano Scientific, USA). Construction and expression of mutants. Based on the sequence and structure analysis of Man113A, alanine scanning was used to determine the contributions of key residues, i.e., Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143, to substrate binding or catalysis. W13F was also constructed to investigate the effects of different residues at this position on catalytic efficiency. Several primer sets (shown in Table S1 in the supplemental material) for site-directed mutagenesis were synthesized. Overlap extension PCR was performed to construct the gene mutants. The expression and purification of the mutants were conducted as described above. Far-UV circular dichroism (CD) spectroscopy was used to determine the secondary structures of Man113A and its mutants. The spectra between 200 and 260 nm were collected at a protein concentration of 1 mg/ml in phosphate-buffered saline buffer (8 g/liter NaCl, 1.44 g/liter Na2HPO4, 240 mg/liter KH2PO4, 200 mg/liter KCl [pH 7.2]) at 25°C by using a MOS-450 CD spectrometer (Bio-Logic, France) equipped with a 1-mm-path-length quartz cuvette and a temperature control device. Each spectrum was an average of three consecutive scans and was corrected by subtracting the buffer spectrum. Comparison of the catalytic properties of wild-type Man113A and its mutants. Specific activities of Man113A and mutants on locust bean gum and konjac flour were measured at a substrate concentration of 10 mg/ml under the standard conditions. The Km, Vmax, and kcat values were determined at pH 6.0 and 50°C for 5 min in 100 mM Na2HPO4-citric acid containing 1 to 20 mg/ml locust bean gum as the substrate. The experiments were carried out three times, and each experiment included three replicates. The data were plotted and calculated according to the Lineweaver-Burk method (14).

RESULTS

Gene cloning and sequence analysis. The full-length man113A gene, 933 bp in length and encoding 310 amino acid residues as well as a termination codon, was amplified from the genomic DNA of Alicyclobacillus sp. A4. The molecular mass and pI value of deduced Man113A were estimated to be 39.2 kDa and 5.2, respectively. A BLAST search of the NCBI database showed that the deduced amino acid sequence of Man113A had the highest identity, 99%, to a hypothetical protein from Alicyclobacillus hesperidum (WP_006446612) and 55% identity to the first functionally verified GH 113 ␤-mannanase, AaManA from A. acidocaldarius Tc-4-1 (ABG77968.1) (7). Moreover, the neighbor-joining phylogenetic tree built from amino acid sequences of several similar proteins and representative ␤-mannanases of GH 5, GH 26, and GH 113 showed that Man113A in this work clustered with the GH

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113 ␤-mannanase AaManA (see Fig. S1 in the supplemental material). The low sequence identity indicated that Man113A is a novel member of family 113. SignalP analysis showed that the deduced Man113A had no signal peptide, indicating that Man113A is an intracellular enzyme, as AaManA is. Sequence alignment of the deduced Man113A and other GH 113 sequences indicated several conserved residues (Fig. 1), and the acid/base catalyst and nucleophile of Man113A, Glu144 and Glu224, respectively, correspond to Glu151 and Glu231 of AaManA. Expression and purification of Man113A. The gene fragment encoding Man113A was expressed in E. coli BL21(DE3)-competent cells. No mannanase activity was detected in the cells of uninduced transformants or of induced transformants harboring the empty plasmid pET-30a(⫹). Recombinant Man113A was purified to electrophoretic homogeneity by Ni2⫹-NTA metal-chelating affinity chromatography. The purified Man113A migrated as a single band on SDS-PAGE with a molecular mass of 39 kDa, which is identical to the calculated value (shown in Fig. S2 in the supplemental material). Characterization of purified recombinant Man113A. Using locust bean gum as the substrate, Man113A displayed an optimal activity at pH 6.0 (Fig. 2a) and had a high activity plateau at pH 5.5 to 7.0 (retaining ⬎90% of its maximal activity). The enzyme was found to be stable over a pH range from 5.0 to 10.0 for 1 h, since it retained ⬎60% of the initial activity after incubation without substrate at 37°C for 1 h (Fig. 2b). Man113A showed mesophilic properties as a typical intracellular hydrolyase, exhibiting the best performance at 55°C (Fig. 2c) and maintaining thermostability at 40°C (Fig. 2d). When incubated at 60°C without substrate, the enzyme lost activity rapidly. The activity of purified Man113A was not affected by most metal ions or chemical reagents at concentrations of 1 and 5 mM (as shown in Table S2 in the supplemental material). It was strongly inhibited by Ag⫹, Cu2⫹, and SDS and moderately enhanced by ␤-mercaptoethanol and Co2⫹. Substrate specificity. Purified Man113A exhibited typical ␤-mannanase activity on mannan polysaccharides containing ␤-1,4-mannosidic linkages (Table 1), while no activity was detected against barley ␤-glucan, birchwood xylan, and CMC-Na. It exhibited the highest activity toward konjac flour and locust bean gum, moderate activity on ivory nut mannan, and varied activities on guar gum with different galactose contents. When using galactomannans with increased galactose contents of 21, 28, 34, and 38% as the substrates, Man113A showed decreased activities of 391.3, 302.8, 36.2, and 34.9 U/mg, respectively. In comparison to its counterparts, Man113A exhibited much lower activity than that of AaManA (1,056 U/mg) toward konjac flour (7) and that of MAN5A from Bispora sp. strain MEY-1 (3,373 U/mg) against locust bean gum (15) but higher activity than that of Man5XZ3 from Aspergillus nidulans XZ3 (184.8 U/mg against locust bean gum) (16). Hydrolysis of mannans and mannooligosaccharides. The hydrolysis profiles of locust bean gum and konjac flour by Man113A varied due to the different compositions of substrates (Fig. 3). The main hydrolysis products of locust bean gum and konjac flour have been reported as galactose-branched mannooligosaccharides (17) and glucose-linked mannooligosaccharides (18). In the case of Man113A, a larger amount of shorter sugars was detected in the hydrolysis products of locust bean gum while higher hydrolytic activities (defined by the total reduced sugar equivalents) were detected when using konjac flour as the substrate. This disparity might be ascribed to the fact that konjac flour contains glucose

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Substrate Recognition of a Novel GH 113 ␤-Mannanase

FIG 1 Multiple-sequence alignment of Man113A and other GH 113 members by using Vector NTI 10.0. Identical and similar residues are shaded black and gray, respectively. The two putative catalytic residues are indicated by asterisks. The conserved regions are boxed.

branches of various lengths and is degraded into relatively larger hybrid oligosaccharides. Both substrates had similar product ratios, and mannobiose and mannotriose, as the main products, accumulated at high rates.

The catalytic efficiencies of Man113A on mannooligosaccharides are summarized in Table 2. The activity order was mannohexaose ⬇ mannopentaose ⬎ mannotetraose ⬎ mannotriose. The kcat/Km values of mannohexaose, mannopentaose, and

FIG 2 Enzymatic properties of purified recombinant Man113A. (a) Effect of pH on Man113A activity; (b) pH stability; (c) effect of temperature on Man113A activity; (d) thermostability. Each value in the panel represents the mean ⫾ the standard deviation (n ⫽ 3).

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TABLE 1 Substrate specificity of Man113A Substrate

Main ingredient (mannose/galactose ratio)

Mean sp act (U/mg) ⫾ SDb

Konjac flour Locust bean gum Ivory nut mannan

Glucomannan (100:0) Galactomannan (80:20) Galactomannan

370.4 ⫾ 11.8 298.8 ⫾ 6.4 105.3 ⫾ 10.1

Guar guma

Galactomannan (79:21) Galactomannan (72:28) Galactomannan (66:34) Galactomannan (62:38)

391.3 ⫾ 4.5 302.8 ⫾ 2.5 36.2 ⫾ 2.2 34.9 ⫾ 4.9

a Galactomannan guar gum was prepared by treatment with ␣-galactosidase to reduce the galactose content. b The specific activity data are shown as means ⫾ SD (n ⫽ 3).

mannotetraose were the same order of magnitude, while that of mannotriose was less than one-tenth that for mannohexaose and mannopentaose. Transglycosylation activity of Man113A. TLC analysis indicated that Man113A exhibited considerable transglycosylation activity, in addition to hydrolytic activity, when a high concentration of mannooligosaccharide (50 mM) was used as the substrate. Man113A had no activity on mannotriose during the first 10 min of incubation but catalyzed transglycosylation afterward to produce mannotetraose and mannopentaose (Fig. 4A). When mannotetrose was used as the substrate, small oligosaccharides were first produced and the final products were mainly mannobiose and mannotriose, as well as a small amount of mannopentaose as the transglycosylation product (Fig. 4B). Similar reaction patterns were observed with mannopentaose (Fig. 4C). However, when

mannohexaose was used as the substrate, hydrolysis instead of transglycosylation predominated over the whole process (Fig. 4D). As a result, Man113A showed hydrolysis rather than transglycosylation in the late stage of incubation with mannotetraose or mannopentaose, an observation similar to that for the previously reported counterpart AaManA (7). In contrast, the endo-␤-1,4mannanases ManA and ManC from A. nidulans FGSC A4 had lower catalytic efficiencies on mannotetraose and mannopentaose, but the transglycosylation products of mannotetraose, i.e., mannopentaose and mannohexaose, accumulated over the 150 min of coincubation (19). These contrasting results indicated different relationships between hydrolysis and transglycosylation for mannanases of different families. Construction and kinetic characterization of Man113A mutants. Site-directed mutagenesis and alanine scanning were performed to identify the functions of the key residues identified as described above. Eight mutants were successfully constructed and expressed in E. coli BL21(DE3). Recombinant enzymes were purified as described above and confirmed by SDS-PAGE (see Fig. S2 in the supplemental material). The secondary structures of Man113A and its mutants were determined by CD spectrometry. Two distinct negative peaks were detected, at approximately 208 and 222 nm (see Fig. S3 in the supplemental material). These characteristic values proved the presence of a predominant ␣-helix structure (20). The CD spectra of Man113A and its mutants showed little variance, indicating that all mutations did not affect the folding structure of the protein. The specific activities and kinetic parameters of wild-type Man113A and eight mutants were determined using locust bean gum and konjac flour as the substrates (data shown in Table 3).

FIG 3 Chromatograms of the products and accumulation of mannooligosaccharides from hydrolysis of locust bean gum (a and b) and konjac flour (c and d) by Man113A. (a and c) Chromatograms 1 to 4, with incubation for 0, 10, 30, and 60 min, respectively. (b and d) Accumulation of hydrolysis products. M1, mannose; M2, mannobiose; M3, mannotriose; M4, mannotetraose; M5, mannopentaose.

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TABLE 2 Comparison of the catalytic efficiencies of wild-type Man113A, its mutants. and the ␤-mannanases of other families using various mannooligosaccharides as substrates kcat/Kmb (/s/mM) GH family and mannasea

M3

M4

M5

M6

Source or reference

GH 113 Man113A N90A* Y292A* C143A* W13F* AaManA

6.54 1.30 7.60 0.48 3.54 ND

29.70 11.37 45.63 6.98 14.80 1.94

72.70 20.34 30.36 29.58 54.48 7.02

75.70 31.42 21.21 16.49 73.76 7.99

This study This study This study This study This study 7

GH 5 AnMan5A AnMan5B BaMan5A†

ND 0.16 8.83 ⫻ 10⫺4

6.00 40.0 2.33

23.00 36.60 2.50 ⫻ 102

1.09 ⫻ 102 61.60 2.00 ⫻ 103

19 23 22

GH 26 CjMan26C† CfMan26A† BsMan26A†

30.00 0.377 2.17 ⫻ 10⫺2

2.33 ⫻ 102 5.98 4.67

ND 46.40 45.00

4.67 ⫻ 102 1.12 ⫻ 102 1.12 ⫻ 102

11 26 22

a b

*, Man113A mutants constructed in the present study; †, data were converted from “/min/␮M” or “/min/M” to “/s/mM.” The catalytic efficiency values were calculated following the formula kcat/Km ⫽ ln([S0]/[St])/E/time, where E represents the concentration of enzyme. ND, no data available.

The Km and kcat values of Man113A for the hydrolysis of locust bean gum were 3.9 mg/ml and 232.1/s, respectively, which were slightly inferior to those of AaManA (2.4 mg/ml and 340/s, respectively) (7). All mutants exhibited dramatic decreases in hydrolytic capacity with quite lower specific activities and catalytic efficiencies. The replacement of hydrophobic aromatic residues Trp13, Trp96, Tyr196, Trp274, and alkaline residue Arg97 located next to the predicted stacking platform Trp96 caused almost complete inactivation of the enzyme, indicating the vital roles of these residues. The mutation of Cys143 might affect the spatial position or side chain of the adjacent nucleophile Glu144, thus accounting for the sharp reduction in catalytic efficiency (kcat/Km value from 59.4 to 1.34 /s/[mg/ml]). This position is strictly occupied by an asparagine in most GH-A enzymes (21). The N90A mutant showed approximately 5- and 2-fold decreases in Km and kcat values, respectively. The Y292A mutant retained only 10% of the catalytic efficiency of Man113A, with a high Km value (36.3 mg/ml) and unchanged kcat. That is, the Y292A mutant had a reduced substrate affinity but retained the cleaving efficiency. The long distance of the product exit from the catalytic center might explain this finding. The W13F mutant retained only one-third of the observed activity of wild-type Man113A, with an unaltered kcat value and a doubled Km value. The catalytic efficiencies of the N90A, Y292A, C143A, and W13F mutants using various mannooligosaccharides as the substrates were also determined (Table 2). The N90A and C143A mutants had much lower kcat/Km values with all tested mannooligosaccharides, whereas the Y292A and W13F mutants displayed different patterns. The Y292A mutant maintained catalytic efficiencies toward mannotriose and mannotetraose similar to those of Man113A but ⬍50% of those of the wild type with mannopentaose and mannohextaose. In contrast, the W13F mutant mainly showed differences in catalytic efficiency toward mannotriose and mannotetraose.

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DISCUSSION

As a newly defined GH family, GH 113 has only one characterized and structure-resolved member, i.e., AaManA from A. acidocaldarius Tc-12-31 (7). AaManA has been reported to be an intracellular ␤-mannanase with higher activity on glucomannan (konjac flour) than on galactomannan (locust bean gum) and relatively low hydrolytic activity on mannooligosaccharides. However, the lack of experimental evidence has limited comprehensive clarification of the biochemical and functional features of GH 113 members. It is necessary to broaden and deepen the knowledge of this family by identifying and investigating more members. In the present study, we identified and characterized another ␤-1,4-mannanase of GH 113, Man113A, which shares certain similarities in sequence and enzymatic properties with AaManA. Man113A exhibits common features of bacterial ␤-mannanases, such as mesophilic and neutral properties and moderate hydrolytic ability. However, it shows a downward trend of activity toward guar galactomannan with an increasing percentage of galactosyl (the ratios are indicated in Table 1), which was not verified for AaManA. The sugar units in mannan branches may give rise to a steric hindrance to the accessibility of Man113A. Another finding is that Man113A also had greater capacity to degrade glucomannan (konjac flour, 370.4 U/mg) than galactomannan (locust bean gum, 298.8 U/mg). This property is similar to that of BaMan5A from Bacillus agaradhaerens, with a higher kcat/Km value on konjac flour (2.6 ⫻ 104/min/␮M) than on locust bean gum (1.1 ⫻ 104/min/␮M) (22). The polar residue Asn127 of BaMan5A at the ⫹2 subsite may contact the 2-OH group of the substrate in either an axial (as in mannose) or an equatorial (as in glucose) configuration. Most fungal ␤-mannanases of GH 5 and GH 26 have higher activity on locust bean gum than on konjac flour, whereas bacterial ␤-mannanases might have relatively looser structures that allow recognition of different substrates. In comparison with ␤-mannanases of other families, Man113A of

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FIG 4 TLC analysis of the transglycosylation products of mannotriose (A), mannotetraose (B), mannopentaose (C), and mannohextaose (D) by Man113A. S, mannooligosaccharide standards; M1, mannose; M2, mannobiose; M3, mannotriose; M4, mannotetraose; M5, mannopentaose; M6, mannohextaose. The reaction time of each sample was indicated at the bottom.

GH 113 is strict with regard to the branch ratio of substrates but adaptable to backbone heterogeneity. Although Man113A had a lower specific activity than AaManA on locust bean gum, the kcat/Km values on mannotetraose through

mannohexaose were much higher. The two GH 113 ␤-mannanases had the same tendency, exhibiting almost identical catalytic efficiencies on mannopentaose and mannohexaose (7.02 and 7.99/s/mM for AaManA and 72.7 and 75.7/s/mM for Man113A,

TABLE 3 Catalytic activities and kinetic parameters of wild-type Man113A and its mutantsa Sp act (U/mg) Enzyme

Locust bean gum

Konjac flour

Km (mg/ml)

kcat (/s)

kcat/Km (/s/[mg/ml])

Wild type W13F Y292A N90A C143A W96A W13A R97A W274A Y196A

298.8 100.0 41.3 24.9 7.9 1.16 0.49 0.25 0.03 ND

370.2 127.7 49.2 40.6 21.2 3.07 1.46 0.51 0.08 ND

3.9 8.4 36.3 18.9 26.9 ND ND ND ND ND

232.1 229.0 246.8 113.5 35.9 ND ND ND ND ND

59.4 27.2 6.8 6.0 1.3 ND ND ND ND ND

a The specific activities were measured at a substrate concentration of 10 mg/ml. The kinetic parameters Km, kcat, and kcat/Km were determined using locust bean gum as the substrate. ND, not determined.

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Substrate Recognition of a Novel GH 113 ␤-Mannanase

FIG 5 Docked complex of Man113A. (a) Overall structure of AaManA in complex with mannohexaose. (b) The stereoview of the catalytic pocket and visualization of substrate-binding mode of Man113A. Mannohexaose (green) and two catalytic glutamates (red) are depicted as sticks. Residues contributed to hydrophobic (white), and polar (light blue) interactions are depicted as lines. The hydrogen bonds are depicted as dashed yellow lines. All oxygen atoms are colored red.

respectively), lower catalytic efficiencies on mannotetraose (half of those on mannopentaose and mannohexaose), and the lowest efficiencies on mannotriose. This illustrated that to carry out efficient hydrolysis, both GH 113 enzymes need at least five subsites, which differs from the requirement of six units for AnMan5A (19). There are some exceptions; for instance, endo-acting AnMan5B (23) hydrolyzed mannotetraose at the same levels with mannopentaose and mannohexaose, and CjMan26C (11) displayed a relatively high kcat/Km value on mannotriose with an exomode cleavage. In addition, among endo-␤-mannanases, Man113A showed considerable catalytic efficiency on mannotriose (6.54/s/mM), even higher than the exo-acting CjMan26C. These data could account for the main hydrolysis products of mannobiose and mannotriose and demonstrated the merit of Man113A to degrade mannans more completely. Apparently, Man113A hydrolyzed substrates in a standard endo-acting mode, which was proved by the relatively high catalytic efficiencies on longer mannooligosaccharides like mannopentaose and mannohexaose and the highest accumulation of mannobiose and mannotriose in mannan hydrolysis. Although the time course of hydrolysis by Man113A was similar to that of an endo-processive mannobiohydrolase, RsMan26H from Reticulitermes speratus (21), its ratios of released mannobiose/(mannose ⫹ mannotriose), i.e., degree of processivity (24), were 1.45 for locust bean gum and 1.17 for konjac flour, respectively. The results suggested that Man113A cleaves sugar chains in a nonprocessive mode. The putative structure of Man113A in complex with mannohextraose was obtained by means of homology modeling and molecular docking. The protein-ligand interactions were predicted and are displayed in Fig. 5. The catalytic residues Glu144 and Glu224 are located at the center of the hydrophobic area formed by aromatic amino acid residues Trp13, Trp96, His176, Tyr196, Tyr238, and Trp240. The substrate mannohextraose occupied the ⫺4 to ⫹2 subsites of this pocket and fixed ⫺1 and ⫹1 of manno-

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hextraose at two sides of Glu144, with an accessible distance of 4.2 Å between the glycosidic bonding oxygen atom (O-19 of the sugar chain) and carbonyl oxygen atom of the nucleophile (OE2 of E144). Hydrogen bonds were also found between OE2 of E144 and two hydroxyl hydrogen atoms of the sugar ring (O-16 and O-23), grappling the substrate at the proper position. Trp13 and Trp240 seemed to serve as the stacking platforms for the sugar units ⫺3/⫺4 and ⫺1/⫺2, respectively, whereas Trp96, Tyr238, and the subsite 1 sugar moiety formed a typical carbohydrate-␲ sandwich structure. Trp13 was found to be replaced by Phe or Tyr in other GH 113 proteins, such as Phe20 in AaManA. Moreover, the substitutive mutation at this site, i.e., W13F, showed only onethird the activity of the wild-type enzyme, with a doubled Km value. This suggested that Trp13 plays a role in the interaction between the protein and the substrate. The lower catalytic efficiencies of W13F against various mannooligosaccharides might be due to weak hydrophobic stacking, which mainly affected the binding of short oligosaccharides. Asn90 combined with subsite ⫺1 and ⫺2 mannosides, which were close to the cleavage site and remained stable to ensure catalysis by forming hydrogen bonds through two hydroxyl hydrogen atoms, O-11 and O-17, with close distances of 3.0 and 3.1 Å, respectively. The assumption that N90 maintains the stability of the substrate and thus ensures that the reaction taking place smoothly is in agreement with the sharp decrease of the kcat/Km value toward both polysaccharides and mannooligosaccharides. Asn90 might play the same role as Asn126 in BaMan5A, recognizing both mannose and glucose units in the process of hydrolyzing glucomannan konjac flour. This position is strictly occupied by an Asn in most GH-A enzymes (25). By means of molecular docking and experimental confirmation, Man113A was proved to possess the eight functionally conserved residues of GH clan A, as AaManA does (7). To date, microbial ␤-mannanases are classed into GH families 5, 26, and 113,

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all belonging to GH clan A. Nevertheless, structural comparisons among representative ␤-mannanases revealed that the substratebinding pockets of different family members differ substantially. One of the distinctions is that ␤-mannanases of different families may have different modes of binding long-chain substrates. As described previously, two important aromatic residues, Trp13 and Trp240, of Man113A (Phe20 and Tyr247, respectively, in AaManA) provide the stacking platform of subunits ⫺1/⫺2 and ⫺3/ ⫺4, respectively. Two equivalent residues, Trp31 and Trp251 of BaMan5A (see Fig. S4A in the supplemental material; PDB code 2WHL) (22), undertook the same responsibility. However, the endo-acting CfMan26A from Cellulomonas fimi (only Trp322 [see Fig. S4B in the supplemental material]; PDB code 2BVT) lacked the former residue, and the exo-acting CjMan26A (see Fig. S4C in the supplemental material; PDB code 2VX6) lacked both (11, 26). Their binding affinities toward the cleaved portion (nonreducing end) of mannan substrates may account for the different hydrolytic patterns. The other obvious distinction is the residues preceding the acid/base catalyst Glu. Cys occupies this position in both GH 113 ␤-mannanases, whereas Asn and His do in GH 5 and GH 26 members, respectively (11, 22, 27–30). The amino acid at this special site may play a vital role in the stabilization of the acid/base catalyst and correction of the substrate conformation. When this cysteine residue was replaced by alanine, the kcat/Km values toward both polysaccharide and mannooligosaccharide substrates decreased substantially. The immanent mechanism of glucosyl recognition is unclear at this time, requiring more structure information. Taken together, GH 113 endo-␤-mannanases are novel ␤-mannanases with both particular enzymatic properties and special structural features. Further investigation is required to clarify their function and the mechanism of substrate recognition. ACKNOWLEDGMENT

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This research was supported by the National High Technology Research and Development Program of China (863 program, 2012AA022208), the National Natural Science Foundation of China (grant 31172235), and the National Science Fund for Distinguished Young Scholars (grant 31225026).

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FUNDING INFORMATION This work, including the efforts of Huiying Luo, was funded by National High Technology Research and Development Program of China (863 program) (2012AA022208). This work, including the efforts of Bin Yao, was funded by National Science Fund for Distinguished Young Scholars (31225026). This work, including the efforts of Huiying Luo, was funded by National Natural Science Foundation of China (NSFC) (31172235).

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Substrate Recognition of a Novel GH 113 ␤-Mannanase

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A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family.

Few members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of...
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