A Novel Glycoside Hydrolase Family 113 Endo-�-1,4-Mannanase fromAlicyclobacillus sp. Strain A4 and Insight into the SubstrateRecognition 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 ofChinaa; 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 andthe 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 transglyco-sylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactoman-nan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to theonly 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, moleculardocking 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 bindingpockets and key residues of �-mannanases of different families indicated that members of GH 113 and GH 5 have more residuesserving as stacking platforms to support �4 to �1 subsites than those of GH 26 and that the residues preceding the acid/basecatalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH113 �-mannanases and distinguishes them from counterparts of other families.

A major component of hemicellulose in the plant cell walls ofsoftwood, plant seeds, and beans is �-1,4-mannan, including

mannan polysaccharides, glucomannan, galactomannan, and ga-lactoglucomannan. It is composed of a backbone of �-1,4-linkedmannose or a combination of glucose and mannose with sidechains of �-1,6-linked galactose residues (1). The mannan-de-grading enzymes have biotechnological applications in variousareas, such as feed manufacturing (2), paper processing (3), andcoffee extract treatment (4). Mannooligosaccharides, the majorhydrolysis products of mannan, are beneficial as animal nutritionadditives due to their potential prebiotic properties (5). The com-plete degradation of mannan polysaccharides into monomers re-quires 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 randomlyhydrolyzes the �-D-1,4-mannopyranoside linkages and plays themajor role in mannan degradation.

�-Mannanases are widely distributed in various organisms, in-cluding bacteria, yeasts, filamentous fungi, and plants. Based onthe amino acid sequence and structural similarities of catalyticdomains, �-mannanases are grouped into three glycoside hydro-lase (GH) families in the Carbohydrate-Active enZYmes (CAZy)database (6), i.e., 5, 26, and 113. GH 113 is a newly defined familythat belongs to GH clan A and shares the same (�/�)8 foldingstructure and catalytic machinery. Two glutamate residues at theC termini of �4 and �7 sheets serve as a general acid/base and anucleophile to catalyze the cleavage of glycosidic bonds via a re-taining double-displacement mechanism. As yet, 180 sequenceshave been classified into GH 113 (http://www.cazy.org/GH113.html); moreover, only one member, AaManA from Alicyclobacil-

lus acidocaldarius Tc-12-31, has been functionally characterizedand its structure resolved (7). AaManA is an intracellular endo-�-1,4-mannanase with transglycosylation activity, and several resi-dues related to substrate binding are identified by sequence align-ments and structure analysis. However, the catalytic mechanismand 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 bacteriumAlicyclobacillus sp. strain A4 (8) and expressed in Escherichia coli.The catalytic properties and residues related to substrate recogni-tion of Man113 were determined by biochemical characterizationand site-directed mutagenesis. We provide here valuable informa-tion 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 novelglycoside hydrolase family 113 endo-�-1,4-mannanase from Alicyclobacillus sp.strain A4 and insight into the substrate recognition and catalytic mechanism ofthis 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, binyao@caas.cn, orPengjun Shi, shipengjun@caas.cn.

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 METHODSStrains, media, vectors, and chemicals. The donor strain Alicyclobacillussp. strain A4 (CGMCC3147) was obtained from the China General Mi-crobiological Culture Collection Center. Escherichia coli Trans1-T1 andthe vector pEASY-T3 from TransGen (China) were used for gene cloningand sequencing, respectively. E. coli BL21(DE3) (TransGen) and the vec-tor pET-30a(�) (Novagen, USA) were used for heterologous protein ex-pression. The DNA purification kit, restriction endonucleases, and LATaq DNA polymerase were purchased from TaKaRa (Japan). The high-fidelity DNA polymerase Fast Pfu was purchased from TransGen. T4DNA ligase was purchased from Promega (USA). Locust bean gum, kon-jac flour, ivory nut mannan, guar gum, barley �-glucan, birchwood xylan,and carboxymethyl cellulose sodium salt (CMC-Na) were obtained fromSigma-Aldrich (USA). Mannooligosaccharide standards (mannose, man-nobiose, mannotriose, mannotetrose, mannopentaose, and manno-hexaose) and high-viscosity guar galactomannans containing 21, 28, 34,or 38% galactose were purchased from Megazyme (Ireland). All otherchemicals were of analytical grade.

Gene cloning and sequence analysis. The �-mannanase geneman113A was amplified from the genomic DNA of Alicyclobacillus sp. A4by using the primers man113EF and man113ER (shown in Table S1 in thesupplemental material), which were designed in accordance with the par-tial genome sequence (data unpublished; the GenBank accession numberfor this gene is KC460333.1). PCR products were purified and ligated intothe pEASY-T3 vector and then transformed into E. coli Trans1-T1 forsequencing. Nucleotide and deduced amino acid sequences were alignedusing 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 weightof the deduced protein, and align multiple sequences. The signal peptidewas predicted by the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/). The neighbor-joining phylogenetic tree based on the deducedamino acid sequences was built using the MEGA software (version 6.0).

Expression and purification of recombinant Man113A. The codingsequence of man113A was amplified by PCR using the primers Man113EFand Man113ER (see Table S1 in the supplemental material), and the PCRproducts were purified as described above. Both the purified PCR prod-ucts and pET-30a(�) were digested by EcoRI and HindIII and ligateddownstream of the His tag coding sequence. The recombinant plasmidswere then individually transformed into E. coli BL21(DE3)-competentcells. The positive transformants were screened and checked by DNAsequencing. Man113 expression in E. coli BL21(DE3)-competent cells wasinduced at 30°C for 6 h by 0.6 mM IPTG (isopropyl-�-D-thiogalactopy-ranoside) at the end of logarithmic growth. Cultures were centrifuged at12,000 � g and 4°C for 5 min. The cells (�5 g) were resuspended in 25 mlof lysis buffer (20 mM Tris-HCl [pH 7.0]) and disrupted by sonication onice with 100 cycles of 5-s short bursts at 200 W and 3 s of cooling by use ofan ultrasonic cell disruptor (Scientz, China). The cell debris was removedby centrifugation, and the supernatant was subjected to Ni2�-NTA chro-matography with a linear gradient of imidazole (2 to 300 mM) in 50 mMTris-HCl– 0.5 M NaCl (pH 7.6). Fractions exhibiting �-mannanase activ-ity were pooled and subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). Elution peaks of a single band with thepredicted size were recovered and concentrated as a purified enzyme so-lution. The protein concentration was determined by a Bradford assaywith bovine serine albumin as a standard.

Assay of the enzymatic activity. �-Mannanase activity was assayed bymeasuring the amount of reducing sugars released from locust bean gumusing the 3,5-dinitrosalicylic acid (DNS) method (9). The standard reac-tion system contained 100 �l of appropriately diluted enzyme sample and900 �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 termi-nated by the addition of 1.5 ml of DNS reagent. After 5 min of boiling inwater, the mixtures were cooled to room temperature and the absorbanceat 540 nm was measured. For the control sample, the recombinant en-

zyme was added after DNS reagent. One unit of �-mannanase activity wasdefined as the amount of enzyme that released 1 �mol of reducing sugarper min under standard assay conditions. Each experiment was per-formed in triplicate.

Biochemical characterization of purified recombinant Man113A.The optimal pH for Man113A activity was determined at 50°C for 10 minover a pH range of 2.0 to 11.0 using the following buffers: 100 mMNa2HPO4-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 andpreincubated at 37°C for 1 h without substrate, and the residual activitieswere measured under the standard conditions (pH 6.0 and 50°C for 10min). The optimal temperature of Man113A was examined at the optimalpH by measuring the enzyme activity over the temperature range of 20 to90°C. The thermostability was investigated by determining the residualenzyme activity after preincubation at 40, 50, and 60°C and the optimalpH without substrate for various time periods. The samples were collectedat 0, 2, 5, 10, 20, 30, and 60 min, respectively. Enzyme activity assays wereperformed under the standard conditions. The enzyme activity ofMan113A was measured in the presence of 1 or 5 mM (final) concentra-tions 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 anyadditive was used as a blank control. Each experiment was performed intriplicate.

Substrate specificity. To investigate the substrate specificity ofMan113A, activity assays were performed under optimum conditionswith 0.5% (wt/vol) locust bean gum (galactomannan, 4:1), konjac flour(glucomannan), ivory nut mannan, guar gum, barley �-glucan, birch-wood xylan, CMC-Na, and guar galactomannan containing 21, 28, 34,and 38% galactose as the substrate. Calculation of the specific activity ofeach substrate used the standard curve corresponding to each monosac-charide component.

Hydrolysis of mannan and mannooligosaccharides. Products ofmannan and mannooligosaccharide hydrolysis by Man113A wereanalyzed using high-performance anion-exchange chromatography(Thermo Fisher Scientific, USA) with a Carbo-Pac PA200 column (3 �mby 250 mm). For mannans, purified recombinant Man113A (3 U) wasincubated in a 1-ml reaction system containing 1% (wt/vol) locust beamgum or konjac flour at 60°C for various durations. For mannooligosac-charides, the experiments were carried out with 1 U/ml enzyme (0.085�M) and a substrate concentration of 30 �M. Samples (500 �l) werewithdrawn at regular intervals, heated in a boiling water bath for 5 min toterminate 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-kDamolecular mass cutoff (Vivascience, USA) to remove the macromole-cules. 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 0.3ml/min. Calibration curves were plotted using �-1,4-mannooligosaccha-rides as standards. The amount of each mannooligosaccharide was calcu-lated based on the peak area of each standard sample. The catalytic effi-ciencies of Man113A and its mutants against mannooligosaccharides werecalculated by fitting the data to the following equation: catalytic efficiency(kcat/Km) ln([S0]/[St])/E/time, where [S0] represents the initial sub-strate concentration, [St] represents the substrate concentration at a spec-ified time during the reaction, and [E] represents the enzyme concentra-tion (10, 11).

Transglycosylation activity of Man113A. The thin-layer chromatog-raphy (TLC) method was used to investigate the transglycosylation activ-ity of Man113A with mannooligosaccharides (mannotriose, mannote-traose, mannopentaose, and mannohexaose) as both donors andacceptors. Each reaction system containing 50 mM individual mannooli-gosaccharide 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 mix-tures were diluted 50� in ddH2O. The mannooligosaccharide standardsand 3 �l of each diluted sample were spotted on an aluminum-coatedsilica 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 platein 5% sulfuric acid (vol/vol), and then heated at 110°C for 5 min.

Homology modeling and molecular docking of Man113A. The ter-tiary structure of the deduced Man113A was homology modeled using theDiscovery Studio v2.5 software (Accelrys, USA) with the crystal structureof AaManA from A. acidocaldarius (PDB code 3CIV, 55% identity) as thetemplate and refined by the embedded energy minimization program andVerify-3D. The molecular docking of modeled Man113A and manno-hexaose was carried out by AutoDock Vina (12) to elucidate the substratebinding interaction. The docking grid, with an appropriate size of 40 Å by40 Å by 40 Å, was set to center the �-carbon atom of Glu144, which waspredicted to be the nucleophile of Man113A. The docked complexes wereanalyzed based on previous studies (7, 13) to select the most reliable bind-ing conformation. Three-dimensional molecular visualization and figurepreparation were performed with PyMOL (version 1.7.2.1; Delano Scien-tific, USA).

Construction and expression of mutants. Based on the sequence andstructure analysis of Man113A, alanine scanning was used to determinethe 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 atthis position on catalytic efficiency. Several primer sets (shown in Table S1in the supplemental material) for site-directed mutagenesis were synthe-sized. Overlap extension PCR was performed to construct the gene mu-tants. The expression and purification of the mutants were conducted asdescribed above.

Far-UV circular dichroism (CD) spectroscopy was used to determinethe secondary structures of Man113A and its mutants. The spectra be-tween 200 and 260 nm were collected at a protein concentration of 1mg/ml in phosphate-buffered saline buffer (8 g/liter NaCl, 1.44 g/literNa2HPO4, 240 mg/liter KH2PO4, 200 mg/liter KCl [pH 7.2]) at 25°C byusing a MOS-450 CD spectrometer (Bio-Logic, France) equipped with a1-mm-path-length quartz cuvette and a temperature control device. Eachspectrum was an average of three consecutive scans and was corrected bysubtracting the buffer spectrum.

Comparison of the catalytic properties of wild-type Man113A andits mutants. Specific activities of Man113A and mutants on locust beangum and konjac flour were measured at a substrate concentration of 10mg/ml under the standard conditions. The Km, Vmax, and kcat values weredetermined at pH 6.0 and 50°C for 5 min in 100 mM Na2HPO4-citric acidcontaining 1 to 20 mg/ml locust bean gum as the substrate. The experi-ments were carried out three times, and each experiment included threereplicates. The data were plotted and calculated according to the Line-weaver-Burk method (14).

RESULTSGene cloning and sequence analysis. The full-length man113Agene, 933 bp in length and encoding 310 amino acid residues aswell as a termination codon, was amplified from the genomicDNA of Alicyclobacillus sp. A4. The molecular mass and pI value ofdeduced Man113A were estimated to be 39.2 kDa and 5.2, respec-tively. A BLAST search of the NCBI database showed that thededuced amino acid sequence of Man113A had the highest iden-tity, 99%, to a hypothetical protein from Alicyclobacillus hesperi-dum (WP_006446612) and 55% identity to the first functionallyverified GH 113 �-mannanase, AaManA from A. acidocaldariusTc-4-1 (ABG77968.1) (7). Moreover, the neighbor-joining phy-logenetic tree built from amino acid sequences of several similarproteins and representative �-mannanases of GH 5, GH 26, andGH 113 showed that Man113A in this work clustered with the GH

113 �-mannanase AaManA (see Fig. S1 in the supplemental ma-terial). The low sequence identity indicated that Man113A is anovel member of family 113. SignalP analysis showed that thededuced Man113A had no signal peptide, indicating thatMan113A is an intracellular enzyme, as AaManA is. Sequencealignment of the deduced Man113A and other GH 113 sequencesindicated several conserved residues (Fig. 1), and the acid/basecatalyst and nucleophile of Man113A, Glu144 and Glu224, respec-tively, correspond to Glu151 and Glu231 of AaManA.

Expression and purification of Man113A. The gene fragmentencoding Man113A was expressed in E. coli BL21(DE3)-compe-tent cells. No mannanase activity was detected in the cells of un-induced transformants or of induced transformants harboring theempty plasmid pET-30a(�). Recombinant Man113A was puri-fied to electrophoretic homogeneity by Ni2�-NTA metal-chelat-ing affinity chromatography. The purified Man113A migrated as asingle band on SDS-PAGE with a molecular mass of 39 kDa, whichis identical to the calculated value (shown in Fig. S2 in the supple-mental material).

Characterization of purified recombinant Man113A. Usinglocust bean gum as the substrate, Man113A displayed an optimalactivity at pH 6.0 (Fig. 2a) and had a high activity plateau at pH 5.5to 7.0 (retaining 90% of its maximal activity). The enzyme wasfound to be stable over a pH range from 5.0 to 10.0 for 1 h, since itretained 60% of the initial activity after incubation without sub-strate at 37°C for 1 h (Fig. 2b). Man113A showed mesophilic prop-erties as a typical intracellular hydrolyase, exhibiting the best per-formance at 55°C (Fig. 2c) and maintaining thermostability at40°C (Fig. 2d). When incubated at 60°C without substrate, theenzyme lost activity rapidly. The activity of purified Man113A wasnot affected by most metal ions or chemical reagents at concen-trations of 1 and 5 mM (as shown in Table S2 in the supplementalmaterial). It was strongly inhibited by Ag�, Cu2�, and SDS andmoderately 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 de-tected against barley �-glucan, birchwood xylan, and CMC-Na. Itexhibited the highest activity toward konjac flour and locust beangum, moderate activity on ivory nut mannan, and varied activitieson guar gum with different galactose contents. When using galac-tomannans 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 coun-terparts, Man113A exhibited much lower activity than that of Aa-ManA (1,056 U/mg) toward konjac flour (7) and that of MAN5Afrom Bispora sp. strain MEY-1 (3,373 U/mg) against locust beangum (15) but higher activity than that of Man5XZ3 from Asper-gillus nidulans XZ3 (184.8 U/mg against locust bean gum) (16).

Hydrolysis of mannans and mannooligosaccharides. The hy-drolysis profiles of locust bean gum and konjac flour by Man113Avaried due to the different compositions of substrates (Fig. 3). Themain hydrolysis products of locust bean gum and konjac flourhave been reported as galactose-branched mannooligosaccharides(17) and glucose-linked mannooligosaccharides (18). In the caseof Man113A, a larger amount of shorter sugars was detected in thehydrolysis products of locust bean gum while higher hydrolyticactivities (defined by the total reduced sugar equivalents) weredetected when using konjac flour as the substrate. This disparitymight be ascribed to the fact that konjac flour contains glucose

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branches of various lengths and is degraded into relatively largerhybrid oligosaccharides. Both substrates had similar product ra-tios, and mannobiose and mannotriose, as the main products,accumulated at high rates.

The catalytic efficiencies of Man113A on mannooligosaccha-rides are summarized in Table 2. The activity order was manno-hexaose � mannopentaose mannotetraose mannotriose.The kcat/Km values of mannohexaose, mannopentaose, and

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.

FIG 2 Enzymatic properties of purified recombinant Man113A. (a) Effect of pH on Man113A activity; (b) pH stability; (c) effect of temperature on Man113Aactivity; (d) thermostability. Each value in the panel represents the mean � the standard deviation (n 3).

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mannotetraose were the same order of magnitude, while that ofmannotriose was less than one-tenth that for mannohexaoseand mannopentaose.

Transglycosylation activity of Man113A. TLC analysis indi-cated that Man113A exhibited considerable transglycosylation ac-tivity, in addition to hydrolytic activity, when a high concentra-tion of mannooligosaccharide (50 mM) was used as the substrate.Man113A had no activity on mannotriose during the first 10 minof incubation but catalyzed transglycosylation afterward to pro-duce mannotetraose and mannopentaose (Fig. 4A). When man-notetrose was used as the substrate, small oligosaccharides werefirst produced and the final products were mainly mannobioseand mannotriose, as well as a small amount of mannopentaose asthe transglycosylation product (Fig. 4B). Similar reaction patternswere observed with mannopentaose (Fig. 4C). However, when

mannohexaose was used as the substrate, hydrolysis instead oftransglycosylation predominated over the whole process (Fig.4D). As a result, Man113A showed hydrolysis rather than trans-glycosylation in the late stage of incubation with mannotetraose ormannopentaose, an observation similar to that for the previouslyreported counterpart AaManA (7). In contrast, the endo-�-1,4-mannanases ManA and ManC from A. nidulans FGSC A4 hadlower catalytic efficiencies on mannotetraose and mannopen-taose, but the transglycosylation products of mannotetraose, i.e.,mannopentaose and mannohexaose, accumulated over the 150min of coincubation (19). These contrasting results indicated dif-ferent relationships between hydrolysis and transglycosylation formannanases of different families.

Construction and kinetic characterization of Man113A mu-tants. Site-directed mutagenesis and alanine scanning were per-formed to identify the functions of the key residues identified asdescribed above. Eight mutants were successfully constructed andexpressed in E. coli BL21(DE3). Recombinant enzymes were pu-rified as described above and confirmed by SDS-PAGE (see Fig. S2in the supplemental material). The secondary structures ofMan113A and its mutants were determined by CD spectrometry.Two distinct negative peaks were detected, at approximately 208and 222 nm (see Fig. S3 in the supplemental material). Thesecharacteristic values proved the presence of a predominant �-he-lix structure (20). The CD spectra of Man113A and its mutantsshowed little variance, indicating that all mutations did not affectthe folding structure of the protein.

The specific activities and kinetic parameters of wild-typeMan113A and eight mutants were determined using locust beangum and konjac flour as the substrates (data shown in Table 3).

TABLE 1 Substrate specificity of Man113A

SubstrateMain ingredient(mannose/galactose ratio)

Mean sp act (U/mg) �SDb

Konjac flour Glucomannan (100:0) 370.4 � 11.8Locust bean gum Galactomannan (80:20) 298.8 � 6.4Ivory nut mannan Galactomannan 105.3 � 10.1

Guar guma Galactomannan (79:21) 391.3 � 4.5Galactomannan (72:28) 302.8 � 2.5Galactomannan (66:34) 36.2 � 2.2Galactomannan (62:38) 34.9 � 4.9

a Galactomannan guar gum was prepared by treatment with �-galactosidase to reducethe galactose content.b The specific activity data are shown as means � SD (n 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) byMan113A. (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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The Km and kcat values of Man113A for the hydrolysis of locustbean gum were 3.9 mg/ml and 232.1/s, respectively, which wereslightly inferior to those of AaManA (2.4 mg/ml and 340/s, respec-tively) (7). All mutants exhibited dramatic decreases in hydrolyticcapacity with quite lower specific activities and catalytic efficien-cies. The replacement of hydrophobic aromatic residues Trp13,Trp96, Tyr196, Trp274, and alkaline residue Arg97 located next tothe predicted stacking platform Trp96 caused almost completeinactivation of the enzyme, indicating the vital roles of these resi-dues. The mutation of Cys143 might affect the spatial position orside chain of the adjacent nucleophile Glu144, thus accounting forthe sharp reduction in catalytic efficiency (kcat/Km value from 59.4to 1.34 /s/[mg/ml]). This position is strictly occupied by an aspar-agine in most GH-A enzymes (21). The N90A mutant showedapproximately 5- and 2-fold decreases in Km and kcat values, re-spectively. The Y292A mutant retained only 10% of the catalyticefficiency of Man113A, with a high Km value (36.3 mg/ml) andunchanged kcat. That is, the Y292A mutant had a reduced substrateaffinity but retained the cleaving efficiency. The long distance ofthe product exit from the catalytic center might explain this find-ing. The W13F mutant retained only one-third of the observedactivity of wild-type Man113A, with an unaltered kcat value and adoubled Km value.

The catalytic efficiencies of the N90A, Y292A, C143A, andW13F mutants using various mannooligosaccharides as the sub-strates were also determined (Table 2). The N90A and C143Amutants had much lower kcat/Km values with all tested mannooli-gosaccharides, whereas the Y292A and W13F mutants displayeddifferent patterns. The Y292A mutant maintained catalytic effi-ciencies toward mannotriose and mannotetraose similar to thoseof Man113A but �50% of those of the wild type with mannopen-taose and mannohextaose. In contrast, the W13F mutant mainlyshowed differences in catalytic efficiency toward mannotriose andmannotetraose.

DISCUSSION

As a newly defined GH family, GH 113 has only one character-ized and structure-resolved member, i.e., AaManA from A. aci-docaldarius Tc-12-31 (7). AaManA has been reported to be anintracellular �-mannanase with higher activity on glucomannan(konjac flour) than on galactomannan (locust bean gum) andrelatively low hydrolytic activity on mannooligosaccharides.However, the lack of experimental evidence has limited compre-hensive clarification of the biochemical and functional features ofGH 113 members. It is necessary to broaden and deepen theknowledge of this family by identifying and investigating moremembers.

In the present study, we identified and characterized another�-1,4-mannanase of GH 113, Man113A, which shares certainsimilarities in sequence and enzymatic properties with AaManA.Man113A exhibits common features of bacterial �-mannanases,such as mesophilic and neutral properties and moderate hydro-lytic ability. However, it shows a downward trend of activity to-ward guar galactomannan with an increasing percentage of galac-tosyl (the ratios are indicated in Table 1), which was not verifiedfor AaManA. The sugar units in mannan branches may give rise toa steric hindrance to the accessibility of Man113A. Another find-ing is that Man113A also had greater capacity to degrade gluco-mannan (konjac flour, 370.4 U/mg) than galactomannan (locustbean gum, 298.8 U/mg). This property is similar to that ofBaMan5A from Bacillus agaradhaerens, with a higher kcat/Km

value on konjac flour (2.6 � 104/min/�M) than on locust beangum (1.1 � 104/min/�M) (22). The polar residue Asn127 ofBaMan5A at the �2 subsite may contact the 2-OH group of thesubstrate in either an axial (as in mannose) or an equatorial (as inglucose) configuration. Most fungal �-mannanases of GH 5 andGH 26 have higher activity on locust bean gum than on konjacflour, whereas bacterial �-mannanases might have relativelylooser structures that allow recognition of different substrates. Incomparison with �-mannanases of other families, Man113A of

TABLE 2 Comparison of the catalytic efficiencies of wild-type Man113A, its mutants. and the �-mannanases of other families using variousmannooligosaccharides as substrates

GH family and mannasea

kcat/Kmb (/s/mM)

Source or referenceM3 M4 M5 M6

GH 113Man113A 6.54 29.70 72.70 75.70 This studyN90A* 1.30 11.37 20.34 31.42 This studyY292A* 7.60 45.63 30.36 21.21 This studyC143A* 0.48 6.98 29.58 16.49 This studyW13F* 3.54 14.80 54.48 73.76 This studyAaManA ND 1.94 7.02 7.99 7

GH 5AnMan5A ND 6.00 23.00 1.09 � 102 19AnMan5B 0.16 40.0 36.60 61.60 23BaMan5A† 8.83 � 10 4 2.33 2.50 � 102 2.00 � 103 22

GH 26CjMan26C† 30.00 2.33 � 102 ND 4.67 � 102 11CfMan26A† 0.377 5.98 46.40 1.12 � 102 26BsMan26A† 2.17 � 10 2 4.67 45.00 1.12 � 102 22

a *, Man113A mutants constructed in the present study; †, data were converted from “/min/�M” or “/min/M” to “/s/mM.”b 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.

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GH 113 is strict with regard to the branch ratio of substrates butadaptable to backbone heterogeneity.

Although Man113A had a lower specific activity than AaManAon locust bean gum, the kcat/Km values on mannotetraose through

mannohexaose were much higher. The two GH 113 �-man-nanases had the same tendency, exhibiting almost identical cata-lytic efficiencies on mannopentaose and mannohexaose (7.02 and7.99/s/mM for AaManA and 72.7 and 75.7/s/mM for Man113A,

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. Thereaction time of each sample was indicated at the bottom.

TABLE 3 Catalytic activities and kinetic parameters of wild-type Man113A and its mutantsa

Enzyme

Sp act (U/mg)

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

(/s/[mg/ml])Locust bean gum Konjac flour

Wild type 298.8 370.2 3.9 232.1 59.4W13F 100.0 127.7 8.4 229.0 27.2Y292A 41.3 49.2 36.3 246.8 6.8N90A 24.9 40.6 18.9 113.5 6.0C143A 7.9 21.2 26.9 35.9 1.3W96A 1.16 3.07 ND ND NDW13A 0.49 1.46 ND ND NDR97A 0.25 0.51 ND ND NDW274A 0.03 0.08 ND ND NDY196A ND ND ND ND NDa 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 thesubstrate. ND, not determined.

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respectively), lower catalytic efficiencies on mannotetraose (halfof those on mannopentaose and mannohexaose), and the lowestefficiencies on mannotriose. This illustrated that to carry out effi-cient 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-actingAnMan5B (23) hydrolyzed mannotetraose at the same levels withmannopentaose and mannohexaose, and CjMan26C (11) dis-played a relatively high kcat/Km value on mannotriose with an exo-mode cleavage. In addition, among endo-�-mannanases,Man113A showed considerable catalytic efficiency on mannotri-ose (6.54/s/mM), even higher than the exo-acting CjMan26C.These data could account for the main hydrolysis products ofmannobiose and mannotriose and demonstrated the merit ofMan113A to degrade mannans more completely. Apparently,Man113A hydrolyzed substrates in a standard endo-acting mode,which was proved by the relatively high catalytic efficiencies onlonger mannooligosaccharides like mannopentaose and manno-hexaose and the highest accumulation of mannobiose and man-notriose in mannan hydrolysis. Although the time course of hy-drolysis by Man113A was similar to that of an endo-processivemannobiohydrolase, RsMan26H from Reticulitermes speratus(21), its ratios of released mannobiose/(mannose � mannotri-ose), i.e., degree of processivity (24), were 1.45 for locust beangum and 1.17 for konjac flour, respectively. The results suggestedthat Man113A cleaves sugar chains in a nonprocessive mode.

The putative structure of Man113A in complex with manno-hextraose was obtained by means of homology modeling and mo-lecular docking. The protein-ligand interactions were predictedand are displayed in Fig. 5. The catalytic residues Glu144 andGlu224 are located at the center of the hydrophobic area formedby 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-

hextraose at two sides of Glu144, with an accessible distance of 4.2Å between the glycosidic bonding oxygen atom (O-19 of the sugarchain) and carbonyl oxygen atom of the nucleophile (OE2 ofE144). Hydrogen bonds were also found between OE2 of E144and two hydroxyl hydrogen atoms of the sugar ring (O-16 andO-23), grappling the substrate at the proper position. Trp13 andTrp240 seemed to serve as the stacking platforms for the sugarunits 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 Tyrin other GH 113 proteins, such as Phe20 in AaManA. Moreover,the substitutive mutation at this site, i.e., W13F, showed only one-third the activity of the wild-type enzyme, with a doubled Km

value. This suggested that Trp13 plays a role in the interactionbetween the protein and the substrate. The lower catalytic efficien-cies of W13F against various mannooligosaccharides might be dueto weak hydrophobic stacking, which mainly affected the bindingof short oligosaccharides. Asn90 combined with subsite 1 and 2 mannosides, which were close to the cleavage site and re-mained stable to ensure catalysis by forming hydrogen bondsthrough two hydroxyl hydrogen atoms, O-11 and O-17, with closedistances of 3.0 and 3.1 Å, respectively. The assumption that N90maintains the stability of the substrate and thus ensures that thereaction taking place smoothly is in agreement with the sharpdecrease of the kcat/Km value toward both polysaccharides andmannooligosaccharides. Asn90 might play the same role asAsn126 in BaMan5A, recognizing both mannose and glucoseunits in the process of hydrolyzing glucomannan konjac flour.This position is strictly occupied by an Asn in most GH-A en-zymes (25).

By means of molecular docking and experimental confirma-tion, Man113A was proved to possess the eight functionally con-served residues of GH clan A, as AaManA does (7). To date, mi-crobial �-mannanases are classed into GH families 5, 26, and 113,

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

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all belonging to GH clan A. Nevertheless, structural comparisonsamong representative �-mannanases revealed that the substrate-binding pockets of different family members differ substantially.One of the distinctions is that �-mannanases of different familiesmay have different modes of binding long-chain substrates. Asdescribed previously, two important aromatic residues, Trp13and Trp240, of Man113A (Phe20 and Tyr247, respectively, in Aa-ManA) provide the stacking platform of subunits 1/ 2 and 3/ 4, respectively. Two equivalent residues, Trp31 and Trp251 ofBaMan5A (see Fig. S4A in the supplemental material; PDB code2WHL) (22), undertook the same responsibility. However, theendo-acting CfMan26A from Cellulomonas fimi (only Trp322 [seeFig. S4B in the supplemental material]; PDB code 2BVT) lackedthe former residue, and the exo-acting CjMan26A (see Fig. S4C inthe supplemental material; PDB code 2VX6) lacked both (11, 26).Their binding affinities toward the cleaved portion (nonreducingend) of mannan substrates may account for the different hydro-lytic patterns. The other obvious distinction is the residues pre-ceding the acid/base catalyst Glu. Cys occupies this position inboth GH 113 �-mannanases, whereas Asn and His do in GH 5 andGH 26 members, respectively (11, 22, 27–30). The amino acid atthis special site may play a vital role in the stabilization of theacid/base catalyst and correction of the substrate conformation.When this cysteine residue was replaced by alanine, the kcat/Km

values toward both polysaccharide and mannooligosaccharidesubstrates decreased substantially. The immanent mechanism ofglucosyl recognition is unclear at this time, requiring more struc-ture information. Taken together, GH 113 endo-�-mannanasesare novel �-mannanases with both particular enzymatic proper-ties and special structural features. Further investigation is re-quired to clarify their function and the mechanism of substraterecognition.

ACKNOWLEDGMENT

This research was supported by the National High Technology Researchand Development Program of China (863 program, 2012AA022208), theNational Natural Science Foundation of China (grant 31172235), and theNational Science Fund for Distinguished Young Scholars (grant31225026).

FUNDING INFORMATIONThis work, including the efforts of Huiying Luo, was funded by NationalHigh Technology Research and Development Program of China (863program) (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 fundedby National Natural Science Foundation of China (NSFC) (31172235).

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