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Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27*

  • Yuka Okazawa
    Affiliations
    Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
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  • Takatsugu Miyazaki
    Footnotes
    Affiliations
    Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
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  • Gaku Yokoi
    Affiliations
    Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
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  • Yuichi Ishizaki
    Affiliations
    Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
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  • Atsushi Nishikawa
    Affiliations
    Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
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  • Takashi Tonozuka
    Correspondence
    To whom correspondence should be addressed: Dept. of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. Tel.: 81-42-367-5702; Fax: 81-42-367-5705
    Affiliations
    Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
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  • Author Footnotes
    * This work was supported in part by a Grant-in-aid for Scientific Research 26660277 (to T. T.) of the Japan Society for the Promotion of Science. This work was performed with the approval of the Photon Factory Advisory Committee (No. 2014G512), the National Laboratory for High Energy Physics, Tsukuba, Japan. The authors declare that they have no conflicts of interest with the contents of this article.
    1 Present address: National Food Research Institute, National Agricultural and Food Research Organization, Tsukuba 305-8642, Japan.
      Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)8-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes.

      Introduction

      Dextran, a polymer of glucose, consists predominantly of α-1,6-glucosidic linkages, and several dextran-hydrolyzing enzymes have been found (
      • Khalikova E.
      • Susi P.
      • Korpela T.
      Microbial dextran-hydrolyzing enzymes: fundamentals and applications.
      ). Isomalto-dextranase (α-1,6-d-glucan isomalto-hydrolase; EC 3.2.1.94) was found in Gram-positive soil bacteria, Arthrobacter globiformis T6 (NRRL B-4425) (
      • Sawai T.
      • Toriyama K.
      • Yano K.
      A bacterial dextranase releasing only isomaltose from dextrans.
      ) and Kitasatospora sp. NRRL B-11411 (formerly known as Actinomadura sp.) (
      • Sawai T.
      • Ohara S.
      • Ichimi Y.
      • Okaji S.
      • Hisada K.
      • Fukaya N.
      Purification and some properties of the isomalto-dextranase of Actinomadura strain R10 and comparison with that of Arthrobacter globiformis T6.
      ). The A. globiformis T6 enzyme (abbreviated as AgIMD)
      The abbreviations used are: AgIMD
      Arthrobacter globiformis T6 isomalto-dextranase
      BcCIT
      Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase
      CBM
      carbohydrate-binding module
      EcYicI
      Escherichia coli YicI
      GH
      glycoside hydrolase family
      hGAL
      human α-galactosidase
      LaMel36A
      Lactobacillus acidophilus α-galactosidase
      pNP-IM
      p-nitrophenyl α-isomaltoside
      Sme
      methionine sulfoxide
      TVAI
      Thermoactinomyces vulgaris α-amylase I
      PDB
      Protein Data Bank
      Ni-NTA
      nickel-nitrilotriacetic acid.
      liberates isomaltose from the non-reducing end of dextran and isomalto-oligosaccharides through a retaining mechanism (
      • Takayanagi T.
      • Okada G.
      • Chiba S.
      Quantitative study of the anomeric forms of isomaltose and glucose produced by isomalto-dextranase and glucodextranase.
      ,
      • Nihira T
      • Mizuno M
      • Tonozuka T
      • Sakano Y
      • Mori T
      • Okahata Y.
      Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance.
      ). AgIMD also catalyzes transglycosylation, and the enzymatic synthesis of some oligosaccharides using AgIMD has been reported (
      • Kim Y.-K.
      • Tsumuraya Y.
      • Sakano Y.
      Enzymatic preparation of novel non-reducing oligosaccharides having an isomaltosyl residue by using the transfer action of isomalto-dextranase from Arthrobacter globiformis T6.
      ).
      The most notable feature of AgIMD is that the primary structure of the enzyme is homologous to those of α-galactosidases and α-N-acetylgalactosaminidases, despite the fact that AgIMDhydrolyzes a polymer of glucose. AgIMD is classified as a member of glycoside hydrolase family (GH) 27 in the CAZy database (
      • Lombard V.
      • Golaconda Ramulu H.
      • Drula E.
      • Coutinho P.M.
      • Henrissat B.
      The carbohydrate-active enzymes database (CAZy) in 2013.
      ). Another enzyme family, GH36, comprises mainly α-galactosidases and α-N-acetylgalactosaminidases. The three-dimensional structures of the two families, GH27 and GH36, share a common structural core (
      • Dagnall B.H.
      • Paulsen I.T.
      • Saier Jr., M.H.
      The DAG family of glycosyl hydrolases combines two previously identified protein families.
      ,
      • Comfort D.A.
      • Bobrov K.S.
      • Ivanen D.R.
      • Shabalin K.A.
      • Harris J.M.
      • Kulminskaya A.A.
      • Brumer H.
      • Kelly R.M.
      Biochemical analysis of Thermotoga maritima GH36 α-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.
      ), a catalytic (β/α)8 barrel domain, and the two families are demonstrated to be evolutionarily related (
      • Rigden D.J.
      Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36, and 66 share a common evolutionary origin with family 13.
      ). It is interesting to note that GH31 also has a similar catalytic (β/α)8 barrel domain, and the three families, GH27, GH31, and GH36, are categorized into clan GH-D. The main GH31 members function as α-glucosidases and α-xylosidases (
      • Lovering A.L.
      • Lee S.S.
      • Kim Y.W.
      • Withers S.G.
      • Strynadka N.C.
      Mechanistic and structural analysis of a family 31 α-glycosidase and its glycosyl-enzyme intermediate.
      ,
      • Okuyama M.
      • Kaneko A.
      • Mori H.
      • Chiba S.
      • Kimura A.
      Structural elements to convert Escherichia coli α-xylosidase (YicI) into α-glucosidase.
      ), but an α-galactosidase that belongs to GH31 has been reported recently (
      • Miyazaki T.
      • Ishizaki Y.
      • Ichikawa M.
      • Nishikawa A
      • Tonozuka T.
      Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.
      ). Therefore, the structure-function relationships of clan GH-D is complicated, and the study of AgIMD is useful for better understanding of clan GH-D.
      In addition to the unique phylogenetic position of the catalytic domain, AgIMD has another intriguing architectural feature belonging to carbohydrate-binding module (CBM) 35. The CBM35 domains have been shown to share a highly similar fold, although there is considerable diversity in the biological functions of the enzymes possessing CBM35 (
      • Montanier C.
      • van Bueren A.L.
      • Dumon C.
      • Flint J.E.
      • Correia M.A.
      • Prates J.A.
      • Firbank S.J.
      • Lewis R.J.
      • Grondin G.G.
      • Ghinet M.G.
      • Gloster T.M.
      • Herve C.
      • Knox J.P.
      • Talbot B.G.
      • Turkenburg J.P.
      • et al.
      Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.
      ,
      • Correia M.A.
      • Abbott D.W.
      • Gloster T.M.
      • Fernandes V.O.
      • Prates J.A.
      • Montanier C.
      • Dumon C.
      • Williamson M.P.
      • Tunnicliffe R.B.
      • Liu Z.
      • Flint J.E.
      • Davies G.J.
      • Henrissat B.
      • Coutinho P.M.
      • Fontes C.M.
      • Gilbert H.J.
      Signature active site architectures illuminate the molecular basis for ligand specificity in family 35 carbohydrate binding module.
      ). We have previously constructed the expression system of AgIMD in Escherichia coli (
      • Tonozuka T.
      • Suzuki S.
      • Ikehara Y.
      • Mizuno M.
      • Kim Y.-K.
      • Nishikawa A.
      • Sakano Y.
      Heterologous production and characterization of Arthrobacter globiformis T6 isomalto-dextranase.
      ), and the enzyme has been crystallized (
      • Akita M.
      • Mizuno M.
      • Tonozuka T.
      • Sakano Y.
      • Matsui H.
      • Hidaka Y.
      • Hatada Y.
      • Ito S.
      • Horikoshi K.
      Crystallization and preliminary x-ray study of isomalto-dextranase from Arthrobacter globiformis.
      ). Here, we determined the crystal structure of AgIMD and compared it with those of GH27, GH31, and other related enzymes.

      Experimental Procedures

       Construction of Expression Plasmids for Wild-type AgIMD and the Mutants

      An expression plasmid, pETG2Dsp−, which is a derivative of pET3a(+), has been previously obtained (
      • Tonozuka T.
      • Suzuki S.
      • Ikehara Y.
      • Mizuno M.
      • Kim Y.-K.
      • Nishikawa A.
      • Sakano Y.
      Heterologous production and characterization of Arthrobacter globiformis T6 isomalto-dextranase.
      ). To facilitate the purification of AgIMD, an expression plasmid of His-tagged AgIMD was constructed. pETG2Dsp− was digested with NdeI and BamHI, and the fragment was ligated into the NdeI-BamHI site of pET28a(+), resulting in plasmid pET28a-AgIMD. Mutant enzymes were generated by site-directed mutagenesis with a QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). To construct expression plasmids for mutants, oligonucleotides 5′-C TGG TAC GAG GAC GGA AGG GCC GCG AAT ATT GGG CAG GTC-3′ (for D207A), 5′-TCC TGG TAC GAG GAC GGA AGG GCC GCG GCC ATC GGG CAG G-3′ (for D207A/N209A); and 5′-GAA GTT TCG CTC GTA GCG CCG CAT ATG TTC-3′ (for M243A) and their complementary strands were used as primers (mutated amino acid residues are underlined). All the constructs used were verified by DNA sequencing.

       Expression and Purification of AgIMD

      E. coli BL21(DE3) cells harboring pET28a-AgIMD were grown in 1 liter of Luria-Bertani (LB) medium containing kanamycin (50 μg ml−1) to A600 = 0.6–0.8, and then induced with isopropyl β-d-thiogalactopyranoside at a final concentration of 0.1 mm; the incubation was then continued for 18 h at 20 °C. The cells were harvested by centrifugation at 4000 × g for 5 min, resuspended in 40 ml of 20 mm imidazole in 20 mm Tris-HCl buffer (pH 8.0), and disrupted by sonication. The cells and supernatants of the cultures were separated by centrifugation at 12,000 × g for 20 min. The supernatant obtained was applied onto a nickel-nitrilotriacetic acid (Ni-NTA)-agarose (Qiagen, Hilden, Germany) column equilibrated with 20 mm imidazole in 20 mm Tris-HCl buffer (pH 8.0). After washing the column with the same buffer, the enzyme was eluted with 100 mm imidazole in 20 mm Tris-HCl buffer (pH 8.0). The active fractions were collected, and the buffer was replaced by 20 mm Tris-HCl buffer (pH 8.0) using an Amicon Ultra-15 centrifugal filter unit (Merck Millipore, Darmstadt, Germany). The His tag was cleaved by thrombin (1 unit per 1 mg of AgIMD) at 20 °C for 16 h and applied onto a Ni-NTA column with the same procedure, and the flow-through fraction was collected. The AgIMD mutants were expressed and purified in a manner identical to that used for the wild-type enzyme.

       Crystallization, Data Collection, and Model Building

      The enzyme was crystallized at 20 °C using the hanging drop vapor diffusion method, where 1 μl of AgIMD (30 mg ml−1) was mixed with the same volume of well solution containing 50 mm sodium acetate buffer (pH 3.6), 16% (w/v) polyethylene glycol 8000, and 50 mm potassium dihydrogen phosphate. For phase determination, the crystal was first transferred for 1 min to a solution containing 50 mm sodium acetate buffer (pH 3.6) and 20% polyethylene glycol 8000, and was then transferred for 48 h to a solution containing 10 mm lead(II) acetate, 50 mm sodium acetate buffer (pH 3.6), and 20% polyethylene glycol 8000. The obtained crystal was transferred to a cryo-solution of 20% glycerol, 50 mm sodium acetate buffer (pH 3.6), and 20% polyethylene glycol 8000. Crystals of AgIMD-isomaltose and AgIMD-panose were obtained by soaking in well solutions containing 30% (w/v) isomaltose and panose, respectively, for a few seconds. The solution containing the ligand also acted as a cryoprotectant. Diffraction data were collected on the beamlines AR-NW12A and AR-NE3A at the Photon Factory (Tsukuba, Japan). All data were processed and scaled using HKL2000 (
      • Otwinowski Z.
      • Minor W.
      Processing of x-ray diffraction data collected in oscillation mode.
      ). The initial phases were calculated from the single wavelength anomalous dispersion data set using the AutoSol program in the PHENIX suite (
      • Terwilliger T.C.
      • Adams P.D.
      • Read R.J.
      • McCoy A.J.
      • Moriarty N.W.
      • Grosse-Kunstleve R.W.
      • Afonine P.V.
      • Zwart P.H.
      • Hung L.W.
      Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard.
      ). The coarse model obtained was applied for the molecular replacement method with MOLREP (
      • Vagin A.
      • Teplyakov A.
      Molecular replacement with MOLREP.
      ) in the CCP4 program suite (
      • Winn M.D.
      • Ballard C.C.
      • Cowtan K.D.
      • Dodson E.J.
      • Emsley P.
      • Evans P.R.
      • Keegan R.M.
      • Krissinel E.B.
      • Leslie A.G.
      • McCoy A.
      • McNicholas S.J.
      • Murshudov G.N.
      • Pannu N.S.
      • Potterton E.A.
      • Powell H.R.
      • et al.
      Overview of the CCP4 suite and current developments.
      ). The model was refined using REFMAC5 in the CCP4 suite, and manual adjustment and rebuilding of the model were carried out using the program COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ). Solvent molecules were introduced using the program ARP/wARP (
      • Langer G.
      • Cohen S.X.
      • Lamzin V.S.
      • Perrakis A.
      Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.
      ). Validation of the structures was performed using the MolProbity server (
      • Chen V.B.
      • Arendall 3rd, W.B.
      • Headd J.J.
      • Keedy D.A.
      • Immormino R.M.
      • Kapral G.J.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MolProbity: all-atom structure validation for macromolecular crystallography.
      ). Figures were prepared using PyMOL, Caver (
      • Chovancova E.
      • Pavelka A.
      • Benes P.
      • Strnad O.
      • Brezovsky J.
      • Kozlikova B.
      • Gora A.
      • Sustr V.
      • Klvana M.
      • Medek P.
      • Biedermannova L.
      • Sochor J.
      • Damborsky J.
      CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.
      ), and LigPlot (
      • Laskowski R.A.
      • Swindells M.B.
      LigPlot+: multiple ligand-protein interaction diagrams for drug discovery.
      ). The data collection and refinement statistics are summarized in Table 1.
      TABLE 1Data collection and refinement statistics
      SAD PbCl2UnligandedIsomaltose complexPanose complex
      PDB codes5AWO5AWP5AWQ
      Data collection
       BeamlinePF AR-NW12APF AR-NW12APF AR-NE3APF AR-NE3A
       Wavelength (Å)0.950071.01.01.0
       Space groupI41I41I41I41
       Cell dimensions
      a = b (Å)123.9123.6123.7123.9
      c (Å)84.284.784.885.3
       Resolution range (Å)50–2.50 (2.59–2.50)
      Values for the highest resolution shells are given in parentheses.
      50–1.44 (1.51–1.44)
      Values for the highest resolution shells are given in parentheses.
      50–2.00 (2.07–2.00)
      Values for the highest resolution shells are given in parentheses.
      50–1.48 (1.53–1.48)
      Values for the highest resolution shells are given in parentheses.
       Unique reflections22,099116,09843,20196,929
       Redundancy7.2 (6.6)
      Values for the highest resolution shells are given in parentheses.
      5.3 (5.0)
      Values for the highest resolution shells are given in parentheses.
      7.4 (7.4)
      Values for the highest resolution shells are given in parentheses.
      7.1 (6.3)
      Values for the highest resolution shells are given in parentheses.
       Completeness (%)99.9 (100)
      Values for the highest resolution shells are given in parentheses.
      100 (100)
      Values for the highest resolution shells are given in parentheses.
      100 (100)
      Values for the highest resolution shells are given in parentheses.
      91.2 (100)
      Values for the highest resolution shells are given in parentheses.
      I/σ(I)56.8 (24.1)
      Values for the highest resolution shells are given in parentheses.
      14.2 (3.6)
      Values for the highest resolution shells are given in parentheses.
      40.0 (13.7)
      Values for the highest resolution shells are given in parentheses.
      47.9 (6.9)
      Values for the highest resolution shells are given in parentheses.
      Rmerge0.060 (0.157)
      Values for the highest resolution shells are given in parentheses.
      0.058 (0.385)
      Values for the highest resolution shells are given in parentheses.
      0.175 (0.345)
      Values for the highest resolution shells are given in parentheses.
      0.071 (0.359)
      Values for the highest resolution shells are given in parentheses.
      Refinement statistics
      Rwork0.1600.1570.161
      Rfree0.1870.1960.187
       Root mean square deviation
      Bond lengths (Å)0.0100.0090.008
      Bond angles (°)1.451.351.33
       Ramachandran plot (MolProbity)
      Favored (%)96.897.197.2
      Allowed (%)3.02.72.8
      Outliers (%)0.20.20
       No. of atoms
      Protein466146454648
      Sugar4657
      Phosphate ion555
      Acetate ion4
      Water877601794
       Average B2)
      Protein20.118.415.9
      Sugar bound to domain A14.413.7
      Sugar bound to domain D27.225.9
      Phosphate ion22.731.523.6
      Acetate ion22.5
      Water32.326.527.7
      a Values for the highest resolution shells are given in parentheses.

       Measurement of Enzymatic Activity and Protein

      The enzymatic activity of AgIMD was measured as described (
      • Tonozuka T.
      • Suzuki S.
      • Ikehara Y.
      • Mizuno M.
      • Kim Y.-K.
      • Nishikawa A.
      • Sakano Y.
      Heterologous production and characterization of Arthrobacter globiformis T6 isomalto-dextranase.
      ). Briefly, the activity of dextran T2000 (GE Healthcare, Chalfont St. Giles, UK) was measured in 100 mm sodium acetate buffer (pH 5.3) for 30 min at 30 °C. Preparation and measurement of the cleavage of p-nitrophenyl α-d-isomaltoside (pNP-IM) was also performed as described (
      • Tonozuka T.
      • Suzuki S.
      • Ikehara Y.
      • Mizuno M.
      • Kim Y.-K.
      • Nishikawa A.
      • Sakano Y.
      Heterologous production and characterization of Arthrobacter globiformis T6 isomalto-dextranase.
      ). The protein concentration was determined by measuring the absorbance at 280 nm, using the molar extinction coefficient (1 mg/ml = 2.487) calculated by the ExPASy ProtParam server. Kinetic parameters were calculated by nonlinear regression analysis using KaleidaGraph (Synergy Software, Reading, PA).

      Results and Discussion

       Overall Structure of AgIMD

      The recombinant AgIMD was expressed in E. coli and affinity-purified by Ni-NTA-agarose chromatography. The N-terminal His tag was then removed by thrombin cleavage, and the protein was crystallized. The crystals belonged to the tetragonal space group I41 with one monomer in the asymmetric unit. The structure was determined using the single wavelength anomalous dispersion technique with a crystal soaked in lead acetate solution. A coarse model of AgIMD was initially built with a 2.5-Å resolution dataset, and the model was further refined using a 1.44-Å resolution dataset (Table 1). The 2FoFc electron density contoured at 1 σ showed continuous density for almost all the main chain atoms, but the N-terminal segment, GSHMATAVTARPGV, was not visible. The Ramachandran plot calculated with the Molprobity server shows that only one residue, Asp-312, was identified as an outlier. Asp-312 is one of the key amino acid residues in the active site, and the electron density for this residue was well defined.
      The structure of AgIMD consists of three domains, designated domain A (residues 21–367), domain C (368–466), and domain D (467–606), as well as an extra loop comprising residues 11–20 in the N terminus (Fig. 1A). Domain A is composed of a (β/α)8-barrel, and domain C is made up of an antiparallel β-structure. This two-domain architecture is commonly found in GH27 enzymes (
      • Ichinose H.
      • Fujimoto Z.
      • Honda M.
      • Harazono K.
      • Nishimoto Y.
      • Uzura A.
      • Kaneko S.
      A β-l-arabinopyranosidase from Streptomyces avermitilis is a novel member of glycoside hydrolase family 27.
      ). An extra structural component (residues 123–178) is present in domain A, and it is here designated subdomain B because of its resemblance to those found in GH13 and GH31 (described below). Domain D has been classified as CBM35 in the CAZy database, and like other CBM35 structures (
      • Montanier C.
      • van Bueren A.L.
      • Dumon C.
      • Flint J.E.
      • Correia M.A.
      • Prates J.A.
      • Firbank S.J.
      • Lewis R.J.
      • Grondin G.G.
      • Ghinet M.G.
      • Gloster T.M.
      • Herve C.
      • Knox J.P.
      • Talbot B.G.
      • Turkenburg J.P.
      • et al.
      Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.
      ,
      • Correia M.A.
      • Abbott D.W.
      • Gloster T.M.
      • Fernandes V.O.
      • Prates J.A.
      • Montanier C.
      • Dumon C.
      • Williamson M.P.
      • Tunnicliffe R.B.
      • Liu Z.
      • Flint J.E.
      • Davies G.J.
      • Henrissat B.
      • Coutinho P.M.
      • Fontes C.M.
      • Gilbert H.J.
      Signature active site architectures illuminate the molecular basis for ligand specificity in family 35 carbohydrate binding module.
      ) domain D is composed of a β-sandwich fold.
      Figure thumbnail gr1
      FIGURE 1Overall structure of AgIMD. A, ribbon model of AgIMD-isomaltose. The N-terminal extra loop (magenta), domain A (yellow), subdomain B (cyan), domain C (blue), domain D (green), and two isomaltose molecules (red) are indicated. B–H, FoFc omit maps contoured at 3 σ level for isomaltose in domain A of AgIMD-isomaltose (B), isomaltose in domain D of AgIMD-isomaltose (C), panose in domain A of AgIMD-panose (D), a molecule modeled as isomaltose in domain D of AgIMD-panose (E), Sme243 in unliganded AgIMD (F), Sme243 in AgIMD-isomaltose (G), and Sme243 in AgIMD-panose (H) are shown.
      A structural similarity search of domain A (including subdomain B), domain C, and domain D was performed using the DALI server (
      • Holm L.
      • Rosenström P.
      Dali server: conservation mapping in 3D.
      ). Domain A shows a significant similarity to the domains of GH27, GH36, GH31, and GH13 enzymes, all of which consist of (β/α)8-barrel folds (Table 2). Among the GH27 enzymes, similarities with β-l-arabinopyranosidases (
      • Ichinose H.
      • Fujimoto Z.
      • Honda M.
      • Harazono K.
      • Nishimoto Y.
      • Uzura A.
      • Kaneko S.
      A β-l-arabinopyranosidase from Streptomyces avermitilis is a novel member of glycoside hydrolase family 27.
      ,
      • Lansky S.
      • Salama R.
      • Solomon H.V.
      • Feinberg H.
      • Belrhali H.
      • Shoham Y.
      • Shoham G.
      Structure-specificity relationships in Abp, a GH27 β-l-arabinopyranosidase from Geobacillus stearothermophilus T6.
      ) were relatively higher than those with α-N-acetylgalactosaminidases and α-galactosidases (
      • Garman S.C.
      • Garboczi D.N.
      The molecular defect leading to Fabry disease: structure of human α-galactosidase.
      ). Domain C is similar to the antiparallel β-sheet domains of various GH family enzymes. The results of the similarity search of domain D were markedly different from those of domains A and C, and other GH27 members do not possess CBM35, as described below.
      TABLE 2Summary of structural similarity search using the DALI server
      EnzymePDB codeCAZy
      Domain C and domain D are not catalytic domains; therefore, the CAZy classification of the catalytic domain of each enzyme is shown.
      Z-scorer.m.s.d.Aligned residuesSequence identity
      Å%
      Domain A
      Geobacillus stearothermophilus T6 β-l-arabinopyranosidase4NX0GH2730.52.730221
      Thermotoga maritima α-galactosidase1ZY9GH3626.03.028117
      Bacillus halodurans putative α-N-acetylgalactosaminidase3CC1GH2725.82.727919
       Human α-galactosidase (hGAL)3S5YGH2725.42.626219
      L. acidophilus α-galactosidase (LaMel36A)2XN0GH3620.73.026810
      E. coli α-xylosidase YicI (EcYicI)1XSJGH3118.83.126810
      T. vulgaris α-amylase I (TVAI)1JI1GH1317.63.92809
      Streptococcus mutans dextranase3VMNGH669.44.32629
      Domain C
      Streptomyces avermitilis β-l-arabinopyranosidase3A21GH2710.51.67929
       Human α-galactosidase (hGAL)4NXSGH279.21.98224
      Thermus sp. maltogenic amylase1GVIGH138.72.47915
      Geobacillus stearothermophilus β-xylosidase1W91GH397.02.77712
      Paenibacillus polymyxa xyloglucanase2YKKGH446.12.97914
      G. stearothermophilus α-galactosidase4FNQGH364.92.87014
      Domain D
      L. monocytogenes putative 3-α-isomaltosyltransferase4KWUGH3120.41.612629
      B. circulans cycloisomalto-oligosaccharide glucanotransferase3WNKGH6620.21.812732
      Ruminiclostridium thermocellum rhamnogalacturonan acetylesterase2W1WCE1219.51.712724
      Amycolatopsis orientalis exo-β-glucosaminidase2VZRGH218.91.812528
      R. thermocellum dockerin type I/putative β-xylosidase2WZ8GH3918.41.812622
      Podospora anserina β-1,4-mannanase3ZM8GH2617.81.712025
      a Domain C and domain D are not catalytic domains; therefore, the CAZy classification of the catalytic domain of each enzyme is shown.

       Interactions with Isomaltose and Panose in the Active Site

      The crystal structure of the AgIMD-isomaltose complex is almost isomorphous with that of the unliganded form, and clear electron density maps (FoFc, 3 σ) were obtained for two isomaltose molecules (Fig. 1, B and C). One of the molecules is seen in the active site, and the other molecule is bound to domain D (Fig. 1A). The active site structure of AgIMD-isomaltose was compared with that of unliganded AgIMD. GH27 enzymes employ a retaining mechanism, and two Asp residues act as catalytic residues (
      • Hart D.O.
      • He S.
      • Chany 2nd, C.J.
      • Withers S.G.
      • Sims P.F.
      • Sinnott M.L.
      • Brumer 3rd, H.
      Identification of Asp-130 as the catalytic nucleophile in the main α-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase.
      ,
      • Guce A.I.
      • Clark N.E.
      • Salgado E.N.
      • Ivanen D.R.
      • Kulminskaya A.A.
      • Brumer 3rd, H.
      • Garman S.C.
      Catalytic mechanism of human α-galactosidase.
      ). The structural homology with GH27 α-galactosidases indicates that Asp-197 and Asp-265 are identified as a nucleophile and acid/base catalyst, respectively. Based on the position of the catalytic residues, isomaltose is bound to subsites −2 and −1 in AgIMD-isomaltose, and the two glucose residues are labeled Glc −2 and Glc −1 (Fig. 2A). The binding of isomaltose is accompanied by conformational changes in Glu-81, Val-242, and Met-243. The side chain of Glu-81 in unliganded AgIMD points away from the active site, whereas atoms OE1 and OE2 of Glu-81 in AgIMD-isomaltose form hydrogen bonds with atoms O6 and O4 of Glc-2, respectively. Also, the main chain atom O of Val-242 is not oriented toward the active site in unliganded AgIMD, whereas in the AgIMD-isomaltose, significant conformational differences of Val-242 and Met-243 are observed, and atom O of Val-242 interacts with atoms O2 of Glc −1 via a water molecule.
      Figure thumbnail gr2
      FIGURE 2Stereo views of the active site of AgIMD-isomaltose. A, active sites of unliganded AgIMD (green) and AgIMD-isomaltose (red and magenta). In AgIMD-isomaltose, hydrogen bonds linking atom O2 of Glc −1 to oxygen atom of Val-242 via a water molecule (red ball) are indicated as red dashed lines. B, comparison of the residues comprising tunnel-1 of unliganded AgIMD (green) and AgIMD-isomaltose (red and magenta). Red dashed line, hydrogen bond; red ball, water molecule. Arrows indicate conformational changes from the unliganded form to the ligand-bound form. The subsite numbers of isomaltose are labeled.
      We also determined the crystal structure of AgIMD complexed with a trisaccharide, panose (Glc-α(1→6)-Glc-α(1→4)-Glc). AgIMD hydrolyzes panose, albeit inefficiently, to produce isomaltose and glucose (
      • Takayanagi T.
      • Okada G.
      • Chiba S.
      Quantitative study of the anomeric forms of isomaltose and glucose produced by isomalto-dextranase and glucodextranase.
      ). Electron density for two ligand molecules was seen in AgIMD-panose (Fig. 1, D and E), and as with AgIMD-isomaltose, one molecule was found in the active site, and the other molecule was located on domain D. In the active site, three glucose residues were modeled at subsites −2, −1, and +1. The glucose residues at subsites −2 and −1 found in AgIMD-panose and those found in AgIMD-isomaltose are well superimposed (Fig. 3A). The obtained structural models of domain D in the AgIMD-panose complex is nearly identical to that observed in the AgIMD-isomaltose complex, because a glucose residue at the reducing end was disordered, and the molecule bound to domain D was modeled as isomaltose (Fig. 1E).
      Figure thumbnail gr3
      FIGURE 3Active site of AgIMD-panose. A, stereo view of the active sites of unliganded AgIMD (green), AgIMD-isomaltose (magenta), and AgIMD-panose (blue). B, two tunnels (tunnel-1 and tunnel-2) are found near the active site of AgIMD-panose. The routes of the tunnels calculated with the program Caver are shown in blue. Red balls inside the tunnels represent water molecules. The panose molecule (Glc +1, Glc −1, and Glc −2) as well as residues Sme243, His-245, and Phe-247 are indicated. C, schematic drawing of the amino acid residues interacting with panose in domain A of AgIMD-panose. White circle, oxygen atom; black circle, carbon atom; gray circle, nitrogen atom; circle with S, sulfur atom; dashed line, hydrogen bond. The subsite numbers are labeled. Phe-156 and Phe-198 are involved in hydrophobic interactions with panose.

       Tunnel Structures

      There are two tunnels, designated tuunel-1 and tunnel-2, near the methionine residue at position 243 in the active site of AgIMD, and several water molecules are found in the tunnels (Fig. 3B). The methionine residue is oxidized based on the interpretation of the electron density map (Fig. 1, F–H), and thus the residue is described as Sme243 in this paper. There is another possibility that residue 243 is present in two conformations simultaneously. However, the distance between an atom in the side chain of the 243th residue and atom N of Ser-200 is 3.0 Å, and also that between the same atom in the side chain of the 243th residue and atom N of Trp-201 is 3.0 Å. The observation suggests that hydrogen bonds are likely to be present between these atoms, and thus the 243th residue is identified as methionine sulfoxide. It is interesting to note that an elaborate conformational change is seen in tunnel-1. The conformational changes of side chains of Ser-200, Sme243, His-245, Phe-247, and the main chain atom O of Pro-244 are induced when the ligand binds to the active site (Fig. 2B). As a result, tunnel-1 is closed in the unliganded form and open in the ligand-bound form.
      It is unclear whether Sme243 was oxidized spontaneously or by x-ray radiation. A GH27 α-galactosidase from Trichoderma reesei showed a 12-fold increase in activity when treated with H2O2 (
      • Kachurin A.M.
      • Golubev A.M.
      • Geisow M.M.
      • Veselkina O.S.
      • Isaeva-Ivanova L.S.
      • Neustroev K.N.
      Role of methionine in the active site of α-galactosidase from Trichoderma reesei.
      ), and Met-258 is considered to be possibly oxidized based on the structure modeling (
      • Golubev A.M.
      • Nagem R.A.
      • Brandão Neto J.R.
      • Neustroev K.N.
      • Eneyskaya E.V.
      • Kulminskaya A.A.
      • Shabalin K.A.
      • Savel'ev A.N.
      • Polikarpov I.
      Crystal structure of α-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.
      ). However, the residue corresponding to Met-258 in T. reesei α-galactosidase is identified as Phe-313 in AgIMD. In a GH26 β-mannanase from the termite Reticulitermes speratus, a methionine sulfoxide residue, Sme85, is located in the active site (
      • Tsukagoshi H.
      • Nakamura A.
      • Ishida T.
      • Touhara K.K.
      • Otagiri M.
      • Moriya S.
      • Samejima M.
      • Igarashi K.
      • Fushinobu S.
      • Kitamoto K.
      • Arioka M.
      Structural and biochemical analyses of glycoside hydrolase family 26 β-mannanase from a symbiotic protist of the termite Reticulitermes speratus.
      ), but the role of Sme243 is obviously different from that of Sme85 in the GH26 β-mannanase, because Sme243 does not form any hydrogen bonds with the ligand molecule (isomaltose or panose) in the ligand-bound form, unlike in GH26 β-mannanase.
      It is also difficult to explain the role of the tunnels of AgIMD. It is unlikely that the row of water molecules present in the tunnels directly interacts with the acid-base catalyst, Asp-265, as the distance between atom OD1 of Asp-265 and the row of the first water molecule is more than 10 Å. Similar tunnel structures have been reported to be observed in GH13 (
      • Aghajari N.
      • Roth M.
      • Haser R.
      Crystallographic evidence of a transglycosylation reaction: ternary complexes of a psychrophilic α-amylase.
      ,
      • Hondoh H.
      • Saburi W.
      • Mori H.
      • Okuyama M.
      • Nakada T.
      • Matsuura Y.
      • Kimura A.
      Substrate recognition mechanism of α-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans.
      ), GH48 (
      • Chen M.
      • Kostylev M.
      • Bomble Y.J.
      • Crowley M.F.
      • Himmel M.E.
      • Wilson D.B.
      • Brady J.W.
      Experimental and modeling studies of an unusual water-filled pore structure with possible mechanistic implications in family 48 cellulases.
      ), and GH68 (
      • Tonozuka T.
      • Tamaki A.
      • Yokoi G.
      • Miyazaki T.
      • Ichikawa M.
      • Nishikawa A.
      • Ohta Y.
      • Hidaka Y.
      • Katayama K.
      • Hatada Y.
      • Ito T.
      • Fujita K.
      Crystal structure of a lactosucrose-producing enzyme, Arthrobacter sp. K-1 β-fructofuranosidase.
      ). These water paths have been proposed to function as a water drain and/or a water reservoir, and the tunnels of AgIMD might have similar roles.

       Comparison of the Catalytic Domains of AgIMD and GH27 α-Galactosidase

      The catalytic domain of GH27 human α-galactosidase (hGAL) is one of the most structurally homologous proteins to that of AgIMD, and its catalytic mechanism has been extensively studied (
      • Garman S.C.
      • Garboczi D.N.
      The molecular defect leading to Fabry disease: structure of human α-galactosidase.
      ,
      • Guce A.I.
      • Clark N.E.
      • Salgado E.N.
      • Ivanen D.R.
      • Kulminskaya A.A.
      • Brumer 3rd, H.
      • Garman S.C.
      Catalytic mechanism of human α-galactosidase.
      ). The structures of the catalytic domains of AgIMD and hGAL were superimposed (Fig. 4A). Although the fold of AgIMD is basically identical to that of hGAL, three components comprising residues 123–178, 204–216, and 276–289 were not found in hGAL, and thus these components are designated here as subdomain B, loop-1, and loop-2, respectively. Subdomain B of AgIMD is located between the third β-strand (β3) and the third α-helix (α3) of the (β/α)8-barrel. A short loop comprising residues 136–151 is present instead in the corresponding position of hGAL, and this loop is described as loop-A in this paper. Loop-1 of AgIMD is present between β4 and α4, and loop-2 of AgIMD is observed as an insertion of α6.
      Figure thumbnail gr4
      FIGURE 4Comparison of domain A and subdomain B of AgIMD and related enzymes. A, stereo view of the Cα backbones of AgIMD-panose (pink and red) and hGAL (PDB code 3HG3; cyan and blue). In AgIMD, subdomain B, loop-1 and loop-2 are shown in red. In hGAL, subdomain B is shown in blue. The pyranose rings at subsite −1 alone are indicated by an arrow. B, stereo view of the superimposition of the Cα backbones of AgIMD-panose (magenta), TVAI-glucopentasaccharide (PDB code 2D0F; green), EcYicI-fluoroxylopyranosyl intermediate (PDB code 1XSK; blue), and LaMel36A-galactose (PDB code 2XN2; orange). The pyranose rings at subsite −1 alone are indicated by an arrow. C–E, comparison of the folds of β3-subdomain B-α3 of AgIMD (C), EcYicI (D), and TVAI (E). The Cα backbones are rainbow-colored from blue to red from the N to the C termini.
      To compare the residues involved in each subsite of AgIMD and those of hGAL, residues interacting with panose (Fig. 3C) and the corresponding residues in hGAL are listed (Table 3). At subsite −1, four residues are conserved between AgIMD and hGAL (Asp-77/92, Tyr-120/134, Arg-261/277, and Asp-312/266), despite the fact that glucose binds at subsite −1 of AgIMD, whereas galactose binds at subsite −1 of hGAL. Residues at subsite −2 of AgIMD are completely different from those of hGAL. At subsite −2 of AgIMD, atoms OE1 and OE2 of Glu-81 directly form hydrogen bonds with O6 and O4 of Glc −2, respectively, and also atom N of Trp-79 directly forms hydrogen bonds with O6 and O5 of Glc −2 (Fig. 3C). The tryptophan residue, Trp-79, appears to be the key residue of subsite −2 and is involved in substrate stacking interactions (Fig. 5A). In contrast, α-galactosidase has been reported to be an enzyme that catalyzes the hydrolysis of galactosyl residues from the non-reducing end of a variety of oligosaccharides and polysaccharides, and thus subsite −2 is unnecessary for hGAL. In fact, the position equivalent to the Glc −2 binding cleft of AgIMD is occupied by residues Cys-142 and Ala-143, which are part of loop-A in hGAL, and therefore no binding cleft for subsite −2 is found in hGAL (Fig. 5B).
      TABLE 3Comparison of residues involved in the subsites of AgIMD and related enzymes
      SubsiteAgIMDhGALEcYicILaMel36A
      −2Asn-47Cys-52
      No corresponding residue was identified.
      No corresponding residue was identified.
      Trp-79Cys-94Phe-277Phe-345
      Ile-80Phe-145Trp-315Arg-377
      Glu-81Gly-144Gln-381Asp-380
      Phe-156Ala-143Trp-380Arg-447
      −1Tyr-40Trp-47Phe-515Trp-340
      Asp-77Asp-92Asp-306Asp-370
      Tyr-120Tyr-134Trp-345Trp-415
      Arg-195Lys-168Lys-414Lys-480
      Val-242Glu-203Ala-465Cys-530
      Sme243Tyr-207Arg-466Ser-531
      Arg-261Arg-277Trp-479Trp-549
      Side chain of Trp-549 in LaMel36A occupies the positions of both Arg-261 and Asp-312 in AgIMD.
      Asp-312Asp-266Asp-511Trp-549
      Side chain of Trp-549 in LaMel36A occupies the positions of both Arg-261 and Asp-312 in AgIMD.
      Catalytic residueAsp-197Asp-170Asp-416Asp-482
      Asp-265Asp-231Asp-482Asp-552
      +1Phe-198Cys-172Phe-417Asn-484
      Asp-207
      No corresponding residue was identified.
      Asp-185Gly-58
      Asn-209
      No corresponding residue was identified.
      Gly-186His-203
      Asp-267Asp-233Glu-516Glu-586
      Trp-285Leu-206Tyr-194Gly-532
      a No corresponding residue was identified.
      b Side chain of Trp-549 in LaMel36A occupies the positions of both Arg-261 and Asp-312 in AgIMD.
      Figure thumbnail gr5
      FIGURE 5Some key residues in the active sites of AgIMD and comparison with those of related enzymes. Stereo views of AgIMD-panose (A), hGAL-melibiose (PDB code 3HG3) (B), EcYicI-fluoroxylopyranosyl intermediate (PDB code 1XSK) (C), and LaMel36A-galactose (PDB code 2XN2) (D). The two catalytic Asp residues (Asp-197 and Asp-265 in AgIMD), residues at subsite −1 (Tyr-40 and Asp-77 in AgIMD), and other key residues are illustrated. Colors: magenta, catalytic Asp residues; cyan, residues at subsite −1; blue, residues at plus subsites; green, other key residues. The subsite numbers of bound saccharides are labeled.
      Residues involved in subsite +1 of AgIMD are also different from those of hGAL. The report of the hGAL-melibiose structure indicated that few interactions with the glucose portion of melibiose have been found (
      • Guce A.I.
      • Clark N.E.
      • Salgado E.N.
      • Ivanen D.R.
      • Kulminskaya A.A.
      • Brumer 3rd, H.
      • Garman S.C.
      Catalytic mechanism of human α-galactosidase.
      ). However, five residues appear to be involved in the binding of Glc +1 in AgIMD. Atom O2 and atom O3 of Glc +1 directly form hydrogen bonds with atom OD2 of Asp-207 and atom NE1 of Trp-285, respectively, suggesting that Glc +1 binds tightly to AgIMD (Fig. 3C). The substrate, isomalto-oligosaccharide, is expected to form a helix-like structure like panose, and Phe-198 is located at the center of the helical spiral of the substrate (Fig. 5A). Side chains of Asp-207, Asn-209, and Trp-285, which are involved in the binding of Glc +1 of panose, are likely to form hydrogen bonds with Glc +1 of isomalto-oligosaccharide either directly or through water molecules. These residues are located at loops uniquely found in AgIMD, loop-1 (Asp-207 and Asn-209), and loop-2 (Trp-285).

       Site-directed Mutagenesis of Residues Unique to AgIMD

      To assess the role of Asp-207, Asn-209, and Sme243, which are unique to AgIMD, alanine mutants D207A, D207A/N209A, and M243A were constructed. The kinetic parameters of wild-type and mutant AgIMD were determined (Table 4). For the small synthetic saccharide, pNP-IM, the Km values of all the mutants increased only slightly (less than 2-fold). The kcat values of D207A and D207A/N209A for pNP-IM decreased but not significantly (0.37- and 0.39-fold, respectively), whereas the M243A mutation drastically affected the kcat value for pNP-IM (8.2 × 10−3-fold). For dextran, the Km values of all the mutants were almost identical to that of wild-type enzyme, and the kcat values decreased significantly (0.02- to 0.06-fold). The ratio of kcat/Km values of wild type:D207A:D207A/N209A:M243A for pNP-IM was 100:25:24:0.4, whereas that for dextran was 100:3.6:5.6:2.1. The results suggest that Asp-207 is important for the catalysis of the polysaccharide, dextran, whereas Sme243 plays a significant role in the catalysis of both oligosaccharides and dextran.

       Comparison of the Catalytic Domains of AgIMD and the GH13, GH31, and GH36 Enzymes

      A search with the DALI server indicated that highly homologous proteins belonging to GH13, GH31, and GH36 are identified as Thermoactinomyces vulgaris α-amylase I (PDB code 2D0F; hereafter TVAI) (
      • Abe A.
      • Yoshida H.
      • Tonozuka T.
      • Sakano Y.
      • Kamitori S.
      Complexes of Thermoactinomyces vulgaris R-47 α-amylase 1 and pullulan model oligosaccharides provide new insight into the mechanism for recognizing substrates with α-(1,6) glycosidic linkages.
      ), E. coli YicI (PDB code 1XSK; EcYicI) (
      • Lovering A.L.
      • Lee S.S.
      • Kim Y.W.
      • Withers S.G.
      • Strynadka N.C.
      Mechanistic and structural analysis of a family 31 α-glycosidase and its glycosyl-enzyme intermediate.
      ), and Lactobacillus acidophilus α-galactosidase (melibiase; PDB code 1ZY9; LaMel36A) (
      • Comfort D.A.
      • Bobrov K.S.
      • Ivanen D.R.
      • Shabalin K.A.
      • Harris J.M.
      • Kulminskaya A.A.
      • Brumer H.
      • Kelly R.M.
      Biochemical analysis of Thermotoga maritima GH36 α-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.
      ), respectively (Table 2). The (β/α)8-barrel domains of these proteins were superimposed, indicating that the backbone folds are similar among these enzymes (Fig. 4B). It is noteworthy that subdomain B of AgIMD shares structural homology with those of TVAI and EcYicI (Fig. 4, C–E), despite the low similarities in their primary structures (∼10%). In GH13 and GH31 enzymes, subdomain B is found between β3 and α3 of the barrel structure, which is the same position in AgIMD. The superimposition of TVAI-glucopentasaccharide, EcYicI-fluoroxylopyranosyl intermediate, and LaMel36A-galactose shows that the position and orientation of the pyranose ring at subsite −1 of AgIMD is almost identical to those of EcYicI and LaMel36A but is different from that of TVAI, and the structural similarity of the active site between AgIMD and TVAI is low (Fig. 4B).
      The active sites of AgIMD, hGAL, EcYicI, and LaMel36A were compared (Fig. 5). In hGAL and LaMel36A, Trp-47(hGAL)–Trp-340(LaMel36A) is located near atom O4 of galactose at subsite −1, and Tyr-40(AgIMD) and Phe-515(EcYicI) are located near atom O4 of Glc −1 in AgIMD and EcYicI, respectively. The finding suggests that this difference is important for the specificity for the C4-epimeric configuration of pyranose. At subsite −1, the residue equivalent to Phe-198 of AgIMD is found in EcYicI (Phe-417). Also, EcYicI possesses an extra domain in the N terminus, and residues in this N-terminal domain (Asp-185 and Tyr-194) appear to participate in subsite +1, and thus, unlike in α-galactosidase, the N-terminal domain of EcYicI may perform a function similar to Asp-207, Asn-209, and Trp-285 of AgIMD. These observations led us to the conclusion that some key residues found in AgIMD, Phe-198 and Asp-207, are found in only GH31 enzymes and are not conserved in GH27 and GH36 enzymes.

       Domain D, CBM35

      One of the most intriguing features of the structure of AgIMD is the presence of domain D at the C terminus. Structural homology analysis using DALI indicated that the highest Z-score was for a CBM35 domain of an uncharacterized protein Lmo2446 from Listeria monocytogenes EGD-e (PDB code 4KWU). This Lmo2446 protein showed the highest homology to a GH31 protein, 3-α-isomaltosyltransferase from Sporosarcina globispora N75 (
      • Hashimoto T.
      • Kurose M.
      • Oku K.
      • Nishimoto T.
      • Chaen H.
      • Fukuda S.
      • Tsujisaka Y.
      Digestibility and suppressive effect on rats' body fat accumulation of cyclic tetrasaccharide.
      ), among the characterized proteins. The second highest Z-score match was for a CBM35 domain of a GH66 protein, cycloisomalto-oligosaccharide glucanotransferase from Bacillus circulans T-3040 (BcCIT) (
      • Suzuki N.
      • Fujimoto Z.
      • Kim Y.M.
      • Momma M.
      • Kishine N.
      • Suzuki R.
      • Suzuki S.
      • Kitamura S.
      • Kobayashi M.
      • Kimura A.
      • Funane K.
      Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase.
      ). An isomaltose molecule was identified in the FoFc omit map of both AgIMD-isomaltose and AgIMD-panose (Fig. 1, C and E), and the two glucose residues are labeled Glc-(a) and Glc-(b) from the non-reducing end (Fig. 6B).
      Figure thumbnail gr6
      FIGURE 6Stereo views of CBM35(AgIMD) and comparison with other CBM35 domains. A, comparison of the Cα backbones of CBM35(AgIMD)-isomaltose (magenta), CBM35(Lmo2446)-glucose (PDB code 4WKU; blue), and CBM35(BcCIT)-IG8 (PDB code 3WNN; orange). Arrows are as follows: black arrow, Glc-(a); red arrow, long loop comprising residues 502–509 in CBM35(AgIMD); blue arrow, calcium-binding site in CBM35(Lmo2446) and CBM35(BcCIT). B, sugar-binding site of CBM35(AgIMD). Colors and symbols are as follows: green, unliganded CBM35(AgIMD); magenta, CBM35(AgIMD)-isomaltose; red dashed line, hydrogen bond; red ball, water molecule. Arrows are as in A. C, sugar-binding site of CBM35(BcCIT). Colors are as follows: gray, residues conserved in CBM35(AgIMD); orange, residues not conserved in CBM35(AgIMD). Arrows are as in A. D, comparison of the Cα backbones of CBM35 and catalytic domains of AgIMD (magenta), Lmo2446 (blue), and BcCIT (orange). CBM35 domains are superimposed. E, surface model of AgIMD-panose. Colors are as in A. Arrows are as follows: blue arrow, catalytic cleft; black arrow, ligand-binding site in CBM35. The potential polysaccharide-binding cleft is shown in a red box.
      Here, we describe the CBM35 domain D of AgIMD complexed with isomaltose as CBM35(AgIMD)-isomaltose. CBM35(AgIMD)-isomaltose was compared with the CBM35 domain of Lmo2446 complexed with glucose (CBM35(Lmo2446)-glucose; PDB code 4WKU) and also compared with that of BcCIT complexed with isomalto-octaose (CBM35(BcCIT)-IG8; PDB code 3WNN) (Fig. 6A). The result revealed that CBM35(AgIMD) is highly homologous to CBM35(Lmo2446) and CBM35(BcCIT), and the positions of their sugar-binding sites are conserved (Fig. 6A). Despite the similarities of the CBM35 structures, CBM35(AgIMD) has some unique features. The most conspicuous feature is that a long loop comprising residues 502–509 is found in CBM35(AgIMD). Atoms OE1 and ND2 of Gln-502 directly form hydrogen bonds with atom O6 of Glc-(a), atom O3 of Glc-(b), and atom O4 of Glc-(b), indicating that Gln-502 is the most critical residue for recognition of the α-1,6-glucosidic linkage (Fig. 6B). In contrast, no equivalent loop and residue were found in CBM35(Lmo2446) and CBM35(BcCIT).
      In CBM35(BcCIT) the second sugar-binding site has been reported, and Tyr-499, Phe-501, Asp-512, and Trp-514 appear to participate in the binding. The corresponding residues in CBM35(AgIMD) are Ser-559, Pro-561, Ala-572, and Gly-574, suggesting that the second sugar-binding site is not conserved in CBM35(AgIMD). Also, most CBM35 structures have one or two calcium-binding sites, whereas no calcium-binding site was found in CBM35(AgIMD). In CBM35(Lmo2446) and CBM35(BcCIT), one conserved calcium-binding site was observed, and two Glu residues (Glu-970 and Glu-972 in Lmo2446(CBM35); Glu-424 and Glu-426 in CBM35(AgIMD)) interact with the calcium ion, whereas the corresponding residues in CBM35(AgIMD) are identified as Pro-473 and Ala-475.
      What is the role of the CBM35 domain D in AgIMD? CBM35(AgIMD), CBM35(Lmo2446), and CBM35(BcCIT) were superimposed and the CBM35 domains, and the catalytic domains are only illustrated in Fig. 6D to clarify the relations between these two domains. Despite the remarkable similarity of the CBM35 domains, the positions of the catalytic domains of AgIMD, Lmo2446, and BcCIT are completely different, suggesting that the CBM35 domains have divergent functions as mentioned previously (
      • Montanier C.
      • van Bueren A.L.
      • Dumon C.
      • Flint J.E.
      • Correia M.A.
      • Prates J.A.
      • Firbank S.J.
      • Lewis R.J.
      • Grondin G.G.
      • Ghinet M.G.
      • Gloster T.M.
      • Herve C.
      • Knox J.P.
      • Talbot B.G.
      • Turkenburg J.P.
      • et al.
      Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.
      ). A surface model of AgIMD shows that there is a cleft, seeming suitable for binding a polysaccharide chain, near the sugar-binding site of CBM35(AgIMD) (Fig. 6E). However, this cleft is disconnected to the catalytic cleft, and thus it is unlikely that a linear polysaccharide chain directly moves from CBM35(AgIMD) to the catalytic cleft. Dextran has been reported to contain α-1,3- and occasionally α-1,2- and α-1,4-branched linkages (
      • Khalikova E.
      • Susi P.
      • Korpela T.
      Microbial dextran-hydrolyzing enzymes: fundamentals and applications.
      ,
      • Vu B.
      • Chen M.
      • Crawford R.J.
      • Ivanova E.P.
      Bacterial extracellular polysaccharides involved in biofilm formation.
      ), and also this polysaccharide is known to function as a component of bacterial biofilm matrix (
      • Vu B.
      • Chen M.
      • Crawford R.J.
      • Ivanova E.P.
      Bacterial extracellular polysaccharides involved in biofilm formation.
      ). Therefore, the polysaccharide chains could adopt complicated structures and tend to be tangled with each other. Although the overall structure is different, a GH16 β-agarase has an extra substrate-binding site, and an unwinding mechanism for agarose chains has been proposed (
      • Allouch J.
      • Helbert W.
      • Henrissat B.
      • Czjzek M.
      Parallel substrate binding sites in a β-agarase suggest a novel mode of action on double-helical agarose.
      ). It is likely that CBM35(AgIMD) could help to unwind the tangled polysaccharide chains, similar as proposed in the β-agarase.
      A BLAST homology search was carried out to elucidate the physiological function of AgIMD, resulting in some bacteria possessing genes homologous to the AgIMD gene. The most homologous genes were the Caci_6974 gene from Catenulispora acidiphila DSM44928 (48% similarity) (
      • Copeland A.
      • Lapidus A.
      • Glavina Del Rio T.
      • Nolan M.
      • Lucas S.
      • Chen F.
      • Tice H.
      • Cheng J.F.
      • Bruce D.
      • Goodwin L.
      • Pitluck S.
      • Mikhailova N.
      • Pati A.
      • Ivanova N.
      • Mavromatis K.
      • et al.
      Complete genome sequence of Catenulispora acidiphila type strain (ID 139908).
      ) and the BN506_02253 gene from Bacteroides cellulosilyticus CAG:158 (45% similarity), although no CBM35 domain was found in Caci_6974 and BN506_02253. A gene encoding GH66 putative endodextranase (Caci_6973 from C. acidiphila) or GH97 putative α-glucosidase (BN506_02255 from B. cellulosilyticus) was found near the putative isomalto-dextranase genes, whereas no gene encoding GH70 putative dextransucrase was identified in these bacteria. Some bacteria preferentially utilize uncommon sugars such as cyclodextrins, which has been proposed to be beneficial for surviving in a competitive environment (
      • Hashimoto Y.
      • Yamamoto T.
      • Fujiwara S.
      • Takagi M.
      • Imanaka T.
      Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.
      ). It is likely that Caci_6974 and BN506_02253 are involved in efficient utilization of dextran as a special energy source. Although some dextranases have been reported to influence the formation of bacterial biofilm (
      • Hayacibara M.F.
      • Koo H.
      • Vacca-Smith A.M.
      • Kopec L.K.
      • Scott-Anne K.
      • Cury J.A.
      • Bowen W.H.
      The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases.
      ), Caci_6974 and BN506_02253 may not participate in the biofilm formation because the organisms do not possess GH70 enzymes. AgIMD is also likely to play a role similar to that of Caci_6974 and BN506_02253, and perhaps CBM35(AgIMD) is an apparatus for accelerating the hydrolysis of the complicated polysaccharide structure.
      CBMs are often found in biomass-degrading enzymes, and engineering of CBMs is expected to be beneficial for the production of biofuels (
      • Lopez-Casado G.
      • Urbanowicz B.R.
      • Damasceno C.M.
      • Rose J.K.
      Plant glycosyl hydrolases and biofuels: a natural marriage.
      ,
      • Gilbert H.J.
      • Knox J.P.
      • Boraston A.B.
      Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules.
      ). Despite the high homology with other CBM35 domains, CBM35(AgIMD) has unique features, such as no calcium-binding site and the presence of a long loop. Thus, our results provide new information for biotechnological engineering of CBMs.

       Conclusions

      The crystal structure of AgIMD was determined. Although the structure most closely resembles GH27 enzymes, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 hGAL. The fold of subdomain B is basically identical to those of GH31 EcYicI and GH13 TVAI. Four residues at subsite −1 in AgIMD are conserved in hGAL, whereas the residues involved in subsites −2 and +1 in AgIMD are completely different from those in hGAL. Site-directed mutagenesis showed that Asp-207 at subsite +1 in AgIMD is important for the catalysis of the polysaccharide dextran, and the corresponding aspartic acid residue, Asp-185, is present in GH31 EcYicI. The structural feature of domain D, CBM35(AgIMD), is highly homologous to those of CBM35(Lmo2446) and CBM35(BcCIT), which are domains present in the GH31 enzyme Lmo2446 and the GH66 enzyme BcCIT, respectively. These observations lead us to the conclusion that AgIMD has some features found in GH31, GH13, and GH66 enzymes, despite the fact the overall structure most closely resembles GH27 enzymes. GH27, -31, -36, and -66 enzymes have been proposed to share a common origin with those of the GH13 family, and the architecture of AgIMD appears to provide such evidence.

      Author Contributions

      T. T., T. M., and A. N. designed and coordinated the study. T. T. wrote the paper. G. Y. prepared the crystal of unliganded AgIMD. Y. O. prepared the crystals of AgIMD-isomaltose and AgIMD-panose and constructed the plasmids encoding D207A, D207A/N209A, and M243A. Y. O. and Y. I. measured the enzymatic activities of wild-type and mutant AgIMD. T. T. and T. M. analyzed the diffraction data and determined the crystal structures. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Hiromi Yoshida, Shigehiro Kamitori, and Akiko Shimizu-Ibuka for help during the synchrotron data collection and Zui Fujimoto for useful suggestions. We also thank Hayashibara Co., Ltd., for providing various sugars.

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