Crystal Structure of Glucansucrase from the Dental Caries Pathogen Streptococcus mutans

J. Mol. Biol. (2011) 408, 177–186

doi:10.1016/j.jmb.2011.02.028 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

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Crystal Structure of Glucansucrase from the Dental Caries Pathogen Streptococcus mutans Keisuke Ito 1 †, Sohei Ito 1 ⁎†, Tatsuro Shimamura 2,3 †, Simone Weyand 4,5 , Yasuaki Kawarasaki 1 , Takumi Misaka 6 , Keiko Abe 6 , Takuya Kobayashi 2,3 , Alexander D. Cameron 4,5 and So Iwata 2,3,4,5 1

Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka 422-8526, Japan 2 Japan Science and Technology Agency, ERATO, Iwata Human Receptor Crystallography Project, Yoshida-Konoecho, Sakyo-ku, Kyoto 606-8501, Japan 3 Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan 4 Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London SW7 2AZ, UK 5 Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire OX11 0DE, UK 6 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8567, Japan Received 8 November 2010; received in revised form 3 February 2011; accepted 8 February 2011 Available online 25 February 2011 Edited by G. Schulz Keywords: Streptococcus mutans; glucansucrase; dental caries; glycoside hydrolase family 70; circularly permutation

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosylenzyme intermediate in subsite − 1. On the other hand, novel structural features were revealed in subsites + 1 and + 2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite + 1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the

*Corresponding author. Laboratory of Food Protein Engineering, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan. E-mail address: [email protected]. † K.I., S.I. and T.S. contributed equally to this work. Abbreviations used: ASase, amylosucrase; BLA, Bacillus licheniformis α-amylase; GH, glycoside hydrolase; GSase, glucansucrase; GTF, glycosyltransferase; PDB, Protein Data Bank. 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

Crystal Structure of Glucansucrase

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transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1–3) glycosidic linkages or soluble glucan with α(1–6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI. © 2011 Elsevier Ltd. All rights reserved.

Sweet is an important favorable taste quality linked to food intake in humans. Sucrose is the most highly consumed sweetener but also frequently causes dental caries.1 According to the World Oral Health Report 2003 released by the World Health Organization, dental caries is a major health problem in most industrialized countries, affecting 60–90% of school children and the vast majority of adults. If left untreated for a long period of time, dental caries, viewed as a “life-style-related disease,” can result in pain, tooth loss, infection and, in some cases, even death by sepsis. Streptococcus mutans is the pathogen that is most closely associated with dental caries.2 Caries formation is initiated when glucan, a sticky glucose polymer produced by S. mutans, forms a biofilm (or dental plaque) on teeth, which then traps oral bacteria, food debris and salivary components. Acids produced by bacteria in the biofilm as a result of fermentation of dietary carbohydrates such as sucrose, fructose and glucose demineralize the tooth surface, leading to dental caries. High-molecularweight sticky glucan synthesized from sucrose by glucansucrases (GSases), which are extracellular enzymes expressed by S. mutans, plays an essential role in the etiology and pathogenesis of dental caries.3 Sticky glucan adheres to tooth surfaces to form dental plaques, which can in turn cause additional infections, periodontal disease and halitosis.4 GSases are members of glycoside hydrolase (GH) family 70.2 They catalyze the formation of glucan with various types of glucosidic linkages, namely, α(1–3), α(1–4) or α(1–6) bonds, from sucrose via transglucosylation reactions. GSases are classified into four different types based on the nature of the glycosidic linkage they catalyze: mutansucrase, dextransucrase, alternansucrase and reuteransucrase, which catalyze α(1–3), α(1–6), α(1–3 and 1–6) and α(1–4 and 1–6) glycosidic linkages, respectively.5 In oral cavities, sticky glucan synthesis by S. mutans involves three extracellular glycosyltransferases (GTFs): GTF-I, GTF-SI and GTF-S.6 GTF-I and GTF-SI are mutansucrases that synthesize mainly insoluble glucan with α(1–3) glycosidic linkages. GTF-S is a dextransucrase that synthesizes predominantly soluble glucan with α(1–6) linkages.5 GSases have a molecular mass of approximately 160 kDa and are composed of three functional regions: the N-terminal variable junction region, the catalytic region and the C-terminal glucan binding

region.5,7 The highly conserved catalytic region is essential for glucan synthesis by GSases. Several inhibitors of S. mutans GSase have been identified.8–10 Structural information on this class of enzyme could facilitate further development of novel inhibitors of GSases that prevent dental caries formation; however, to date, this type of information has been lacking. Here, we present the crystal structure of GTF-SI, a GH family 70 mutansucrase that synthesizes mainly α(1–3)-linked insoluble glucan. We also report the structures of GTF-SI in complex with the inhibitor acarbose and acceptor maltose. These structures provide critical insight into glucan formation by this type of enzyme. Overall structure of GTF-SI Recombinant and selenomethionine-substituted GTF-SI from S. mutans was expressed, purified and crystallized.11 The detailed experimental procedure for each structure is provided in Supporting Information Materials and Methods. The final refinement statistics for all structures are summarized in Table 1. The monomeric structure of substrate-free GTF-SI is shown in Fig. 1a. While GTF-SI formed a tetramer in the crystal (Fig. S1a), the results of size-exclusion chromatography indicated that GTF-SI is likely a monomer in solution (Fig. S1b), and the structures of the individual tetramer members were almost identical. The current model, refined at 2.1 Å resolution, contains amino acid residues 244–1087 because the crystallization was not succeeded from the entire protein of GTF-SI. The N-terminal His-tag and some of the C-terminal residues (amino acid residues 1088–1163) included in the current expression construct were not observed in the electron density map. The secondary structure of GTF-SI is shown schematically in Fig. S2. The structure of GTF-SI comprised four separate domains: A, B, C and IV (Fig. 1a). Each domain, with the exception of domain C, was composed of two separate regions of the polypeptide chain. Domain IV consisted of IV1 (residues 244–372) and IV2 (residues 1057–1087); domain A consisted of A1 (residues 443–692) and A2 (residues 829–961); and domain B consisted of B1 (residues 373–442) and B2 (residues 962–1056). Domain C was composed of a single stretch of amino acid residues (693–828). GH family 13 amylases have high level of similarity in overall structures, especially in domain

Crystal Structure of Glucansucrase

179

Table 1. Crystallographic statistics Data collection Space group Cell dimensions (Å) Wavelength (Å) Resolutiona (Å) Unique reflections Completenessa I/σa Redundancy Rmergea (%) Refinement No. of protein residues No. of water molecules Rworka (%) Rfreea (%) Average B-factor (Å2) Protein Substrate Solvent rmsd from ideal values Bonds (Å) Angles (°) Ramachandran analysis (%) Most favored Allowed a

Selenomethionine peak

Wild type

Maltose

Acarbose

P21212 a = 295.43 b = 214.50 c = 219.52 0.9792 30.00–2.70 (2.80–2.70) 376,846 99.4 (99.4) 15.2 (2.7) 5.2 9.7 (39.1)

P21212 a = 293.88 b = 215.42 c = 218.95 1.0000 50.00–2.10 (2.18–2.10) 753,230 100.0 (99.9) 22.7 (7.8) 7.0 7.7 (41.7)

P21212 a = 295.42 b = 213.94 c = 220.98 0.9796 50.67–3.09 (3.27–3.09) 248,179 98.2 (99.4) 9.8 (2.3) 3.6 8.6 (35.6)

P21212 a = 295.52 b = 214.41 c = 220.66 0.9796 61.77–3.11 (3.29–3.11) 238,131 95.7 (98.1) 8.2 (2.1) 3.7 9.1 (40.1)

6752 4447 20.3 (21.2) 23.6 (25.3)

6630 229 21.2 (29.7) 24.2 (34.4)

6630 246 21.1 (31.0) 24.4 (34.1)

19.2 — 35.1

46.6 30.0 39.6

45.4 30.0 40.5

0.030 2.27

0.022 1.97

0.020 1.92

88.5 11.5

87.1 12.9

84.6 15.3

Numbers in parentheses refer to the highest-resolution shell.

A. Therefore, the GTF-SI structure was compared with that of Bacillus licheniformis α-amylase (BLA) as a representative example of GH family 13 amylases, while the sequence identity between GTF-SI and BLA is less than 20%. The overall fold of the catalytic domains A, B and C of GTF-SI was similar to that of BLA (Fig. 1a).12 Domain A formed the core of the GTF-SI catalytic domains.5,13 This domain shares a TIM-barrel motif with GH family 13 α-amylases14 but has two additional helices: the α4′-helix (residues 592–602) and the α4″-helix (residues 614–627) (Fig. 1a and Fig. S2). In a previous study, sequence analysis predicted that the secondary structural elements of GH family 70 GSases are circularly permuted as compared to those of GH family 13 αamylases,15 and this was confirmed by the current structure of GTF-SI (Fig. 1a and b). The first helix (α1) of the GTF-SI TIM-barrel was superimposable on the third α-helix (α3) of BLA. The two additional helices, α4′ and α4″, which are not present in the BLA structure, were positioned between the β4strand and the α5-helix of the GTF-SI TIM-barrel (Fig. 1c and Fig. S2). The observed permutations extended to domains B and C. Domain B, which formed a twisted antiparallel sheet of six β-strands, and domain C, which was composed of eight βstrands with a Greek key motif (Fig. 1a), are well conserved among GH family 13 enzymes. 12,16 Domain B, which in BLA is positioned between the β3-strand and the α3-helix, was composed of two

separate polypeptide chains, one located at the Nterminus and the other, at the C-terminus of the TIM-barrel in GTF-SI (Fig. 1b and Fig. S2). Domain C, which is located at the C-terminal end of the BLA TIM-barrel, was positioned between the α6-helix and the β6-strand in GTF-SI. Naturally occurring circular permutations between homologous proteins have been reported previously, but cases of such extensive permutation are very rare.17 As a result of the structural similarity search by the Dali server,3 domain B of GTF-SI is most similar to that of BLA [Protein Data Bank (PDB) code 1VJS] with a Z-score of 2.7. Domain C of GTF-SI is also most similar to that of the BLA part of chimera enzyme (PDB code 1E3X) with a Z-score of 9.2. The correlation between these domains and reaction specificities is not clear because BLA hydrolyzes the α(1–4) glycosidic linkage. Many other structures of GH family 70 GSases may be required to show the role of these domains.18 Domains IV and V are unique to GH family 70 GSases.5 In the GTF-SI structure, domain IV was positioned next to domain B (Fig. 1a). Domain V is thought to be composed of sequences upstream of domain IV1 and/or downstream of domain IV2, which implies that it is located next to domain IV. Although domains IV and V are not essential for the enzymatic activity of GSases,19 domain IV may serve as a “hinge,” swinging glucan-binding domain V toward and away from the catalytic domains to facilitate glucan extension. The three-

180 Crystal Structure of Glucansucrase

Fig. 1. Structure of GTF-SI from the dental caries pathogen S. mutans. (a) Ribbon diagrams of GTF-SI and BLA. In GTF-SI, domains IV, A and B are composed of chains IV1 (orange) and IV2 (salmon), chains A1 (blue) and A2 (purple) and chains B1 (green) and B2 (yellow green), respectively. In BLA, domain B is composed of a single polypeptide chain, while domain A is composed of two chains, as in GTF-SI. Domains C (magenta) of both enzymes are composed of a single polypeptide chain. β-Sheets in the TIM-barrels are numbered as they occur in the amino acid sequence. Bound calcium (red) and sodium (black) ions are shown as spheres. (b) Circular permutation of domains between GTF-SI and BLA. (c) Structure of α-helices α4′ and α4″ of GTF-SI. Acarbose in the active site is shown in a wire frame model. GTF-SI, BLA and ASase are colored red, green and blue, respectively.

Crystal Structure of Glucansucrase

dimensional structure of domain IV seems to be unique (Fig. 1a) because no structural homologues of domain IV were found in the PDB on the Dali server. The structure of domain V was not resolved in the current study. A strong electron density (8 σ) was observed at the interface of domains A and B (Fig. 1a). This was assigned to a Ca2+ based on its similarity to the Ca-binding site I of BLA.12 Substrate-binding site structure: Comparison with amylosucrase Similar to GSases, Neisseria polysaccharea amylosucrase (ASase), which is a GH family 13 amylase, can synthesize an amylose-like polymer from sucrose.20 GTF-SI synthesizes predominantly α(1–3) glucan from sucrose, whereas ASases synthesize α(1–4) glucan from the same substrate. GSases and ASases are believed to share a similar transglycosylation mechanism.5,21,22 The glucosyl and fructosyl moieties of the “primary sucrose” molecule bind to subsite −1 and subsite +1 of the enzyme, respectively. A proton released from glutamate (Glu515 for GTF-SI) attacks the glucosidic oxygen. The bound sucrose is then hydrolyzed, leaving a glycosyl moiety covalently bound to the aspartic acid residue (Asp477 for GTFSI) as an intermediate, which is stabilized by another aspartic acid residue (Asp588 for GTF-SI). After fructose is released from subsite +1, the glucosyl moiety of a “second sucrose” binds to subsite +1. A glutamate residue (Glu515 for GTF-SI) pulls off the proton from the hydroxyl group of acceptor sucrose to produce the activated hydroxyl ion. The hydroxyl ion engages in nucleophilic attack of the glycosylenzyme intermediate in subsite −1 to form a glucan. The structure of ASase from N. polysaccharea was reported by Jensen et al.20 Comparison of the GSase and ASase structures, despite limited sequence identity, revealed common components involved in the transglycosylation in both enzymes. We first investigated the structure of GTF-SI in complex with acarbose, which was resolved at 3.1 Å resolution (Fig. 2a and Fig. S3). Acarbose is a pseudotetrasaccharide and a strong inhibitor of GSase.8 The Ki of acarbose for S. mutans GSase is 0.2. Figure 2a shows the substrate-binding site structure of GTF-SI in complex with acarbose. The structure of the complex was identical with the high-resolution structure of apo GTF-SI (Fig. S4). The clear density corresponding to acarbose (Fig. S3a and b) allows unambiguous modeling of the inhibitor. There were no other binding sites of acarbose in the present molecule. Figure2a also shows the position of the glucosyl moiety in the structure of the covalent glucosyl-enzyme intermediate of the inactive mutant form of ASase superimposed onto the GSase structure. The terminal pyranosyl ring of acarbose in GTF-SI was closely superimposed on the glycosyl intermediate in subsite − 1 in the ASase structure. By

181 analogy to GH family 13 enzymes, it has been proposed that three conserved acidic residues of GTF-SI (Fig. 3), Asp477, Glu515 and Asp588, act as a nucleophile, a general acid/base catalyst and a stabilizer of the glucosyl intermediate, respectively, as described above.5 In addition to these catalytic residues, Arg475 and His587 (GTF-SI numbering) in subsite − 1 are well conserved in family 70 enzymes and family 13 enzymes; Asp909 and Tyr916 are also conserved between family 70 enzymes and ASase. The active-site structure among the GH family 13 members is well conserved. Thus, the amino acid residues such as Arg475, Asp477, Glu515, His587, Asp588 and Tyr916 constructing subsite − 1 of GTFSI are also conserved with those of other GH family 13 amylases, Aspergillus oryzae α-amylase (PDB code 7TAA) and Thermoactinomyces vulgaris R-47 α-amylase 2 (PDB code 3A60). Our results indicated that recognition of the glucosyl moiety of the primary sucrose and formation of the glycosyl-enzyme intermediate at subsite − 1 are well conserved among GH family 70 and 13 enzymes. In contrast, residues located in subsite + 1 of GTFSI, such as Tyr430, Leu433 and Trp517, are not conserved in ASase. Interestingly, GSases and ASase have longer insertion sequences between the β4strand and the α5-helix. Overall, the structure of ASase was comparable to that of GTS-SI; however, the structure between the β4-strand and the α5-helix was quite different (Fig. 1c). The insertions are located adjacent to subsites + 2 and + 3 and seem to be key factors in transglycosylation specificity.20 In addition, the subsite + 1 structure of GTF-SI is not conserved for those of A. oryzae α-amylase (PDB code 7TAA) and T. vulgaris R-47 α-amylase 2 (PDB code 3A60) as for ASase. The use of acarbose to inhibit the GSase activity of S. mutans also results in the loss of activity of maltase-glucoamylase in the small intestine, causing hypoglycemia.23 However, there is no similarity of acarbose recognition between GTF-SI and maltase-glucoamylase (PDB code 2QMJ) because the maltase-glucoamylase belongs to GH family 31. The Ca-binding site in close proximity to subsite + 1 was observed at the interface of domains A and B (Figs. 1a and 2). This site was composed of the side chains of Glu431, Asp437, Asp959 and the mainchain carbonyl oxygen atom of Asn481. The Asn481 side chain formed a hydrogen bond with the C4 and C6 hydroxyl groups of the acceptor maltose (Fig. 2 and Fig. S3c). Thus, the Ca2+-binding site appeared to be essential for the formation or stabilization of subsite + 1. Transglycosylation specificity: Comparison with other GSases To investigate the structural basis of the transglycosylation specificity of GSases in more detail, we

Crystal Structure of Glucansucrase

182 resolved the structure of GTF-SI in complex with maltose at 3.1 Å resolution (Fig. 2b). The structure of the complex was identical with the high-resolution

structure of apo GTF-SI (Fig. S4) with a clear density corresponding to maltose (Fig. S5a and b), which allowed unambiguous modeling of the molecule.

Fig. 2 (legend on next page)

Crystal Structure of Glucansucrase

183

Fig. 3. Primary structural alignment of three GSases from S. mutans and analysis of sequence diversity using the Pfam protein family database. Amino acid residues comprising sugar-binding subsites −1, +1 and +2 in GTF-SI are colored red. The data for these logos consisted of 31 sequences for family 13 enzymes and 12 sequences for family 70 enzymes. The overall height of the stack indicates the sequence conservation at that position, while the height of the symbols within the stack indicates the relative frequency of each amino acid at that position. A few sequences with rare insertions were removed for convenience. Amino acids are colored according to their chemical properties: polar amino acids are green; basic, blue; acidic, red; and hydrophobic amino acids are black.

There were no other binding sites of maltose in the present molecule. Maltose is a well-known transglycosyl acceptor for GSases, but not a donor.24 When maltose serves as an acceptor, the acceptor products with both GTF-S and GTF-I were panose and a series

of homologues of panose linked to the nonreducingend glucosyl unit of maltose by an α(1–6) linkage.24 Indeed, in subsite + 1, the C6 hydroxyl group formed hydrogen bonds with the carboxyl group of Glu515 (Fig. 2b and Fig. S5c), which has been proposed to

Fig. 2. Substrate-binding site structures of GTF-SI. (a) Stereo view of the substrate-binding site with acarbose. (b) Stereo view of the substrate-binding site with maltose. (c and d) Proposed sucrose binding modes to clarify the reaction mechanism of GTF-S, GTF-SI and GTF-I. Acarbose, maltose and sucrose are shown as stick models. Carbon and oxygen atoms in the models are shown in yellow and red, respectively. A heptacoordinated Ca2+ and water molecules are shown in sphere models and colored red and cyan, respectively. The glucosyl intermediate in the structure of ASase is superposed on the GTF-SI structure and is shown in gray. Domains are colored as in Fig. 1a. Green broken lines indicate the putative interactions between sucrose and Asp593. Computational models of binding sucrose were constructed using the programs Coot and Molfeat (FiatLux Co., JPN). The central position of glucosyl unit in subsite +1 was fixed to that of bound maltose.

184 serve as a general acid/base catalyst. Bound maltose, adopting a low-energy chair–chair conformation, was found in the acceptor-binding site composed of subsite + 1, subsite + 2 and a part of subsite + 3 (Fig. S5c). This binding mode of maltose is inconsistent with the preference of GTF-SI for the C3 hydroxyl group using the sucrose as substrate. Fructosyl unit is more bulky than glycosyl unit; therefore, we presumed that the binding mode of sucrose is different to that of maltose, as shown in Fig. 2c and d. Asp593 might interact with the sucrose directly and might interact with maltose via water molecules, although water molecules have not been identified in the ligand-bound structure of GTF at limited resolution. As described later, we concluded that Asp593 is the only residue that influences the type of glucan products produced by GTF-S, GTF-SI and GTF-I. In any case, the proper hydroxyl group was oriented toward the active center, where the C1 carbon of the covalent glucosyl-enzyme intermediate in ASase is located. The glucose moiety in subsite + 1 also interacted with Asn481, Tyr430 and Trp517 (Fig. 2b and Fig. S5c). Trp517 provided the platform for the acceptor glycosyl moiety, and Tyr430 participated in hydrophobic interactions with carbon atoms of the glycosyl moiety in subsite +1. Asn481, which formed part of the Ca2+-binding site (discussed above), participated in hydrogen bonding with the C4 and C6 hydroxyl groups of the glucosyl moiety in subsite +1. Thus, these residues appeared to be critical for the recognition of the moiety in subsite +1. To further investigate the mechanism of glycosyl bond formation, we compared the amino acid sequences of the GH family 70 enzymes (Fig. 3). GTF-SI and GTF-I are mutansucrases that synthesize mainly insoluble glucan with α(1–3) glycosidic linkages. GTF-S is a dextransucrase that synthesizes predominantly soluble glucan with α(1–6) linkages.5 Most of the amino acid residues located in subsites + 1 and + 2 (Fig. 2 and Fig. S2) were conserved in the three enzymes, with the exception of Glu431 and Asp593. This suggested that the glycosyl linkage product is determined by limited differences among the enzymes. The side chains of Glu431 formed part of the Ca-binding site, and Glu431 was in a position opposite the active center. The position of Asp593 in the α4′-helix appeared to be the most critical point for acceptor sugar orientation. Shimamura et al. reported that replacement of Thr at this position in GTF-S with Asp or Glu promotes the synthesis of insoluble glucan, likely having more α(1–3) linkages, while replacement of this Asp residue in GTF-I with Thr promotes soluble glucan synthesis,25 as the soluble glucan likely has more α(1–6) linkages. Moreover, Monchois et al. also reported that mutation of this position in GTF-I of Leuconostoc mesenteroides influences the structure and the size of

Crystal Structure of Glucansucrase

the resultant glucan.26 Our results were consistent with these previous results and suggested that the distinct reaction specificities of GTF-SI and GTF-S are due to structural different structures in subsites +1 and +2, particularly Asp593. In the GTF-SI– maltose structure, the acceptor sugar was sandwiched by Trp517 and Tyr430, and the C6 hydroxyl group of the glucosyl moiety in subsite +1 was pointed toward the active center (Fig. 2b). This result and the preference of GTF-SI for the C3 hydroxyl group using the sucrose indicate that subsites +1 of GSases are capable of accepting the glucose in a dual manner, and a residue, position 593, is the key to determine the type of glucan products. The unique structural features of family 70 enzymes in the α4′- and α4″-helices were observed in the current crystal structure, as described above (Fig. 1c). The results of random mutagenesis of a GSase from another oral streptococci, Streptococcus oralis (47% amino acid identity with GTF-SI) indicated that the N-terminus of the α4′-helix and the adjacent loop stabilize the transition state and determine the specificity of GSases. 27 The Nterminal end of the α4′-helix and the loop between the β4-sheet and the α4′-helix could interact with longer or branched substrates, and modifications in this region could alter the reaction specificity of GSases, including regioselectivity and chain length of the resultant oligosaccharides.25,27 In addition to its role as the glycosyl acceptor, maltose also is a very weak inhibitor for GSase, while the effectiveness is about 1 order of magnitude smaller than that of the acarbose.8 The structure of GTF-SI in complex with maltose revealed that maltose serves as a competitive inhibitor of the second acceptor sucrose (Fig. 2b). This information should prove to be useful in the design of novel GSase inhibitors. Acarbose contains a nonhydrolyzable nitrogen-linked bond that blocks the catalytic activity of various GHs, including maltase-glucoamylase in the small intestine.28 This mechanism could underlie some of the side effects of this inhibitor, such as hypoglycemia.23 New inhibitors that specifically target subsites + 1, + 2 and + 3 of GSase can now be designed based on the structure of the GTF-SI–maltose complex reported herein. Most recently, after the submission of this article, the crystal structure of the enterobacterial GSase GTF-180, a homologue to GTF-SI, was reported by Vujicic-Zagar et al.29 While GTF-180 catalyzes both α(1–6) and α(1–3) glycosidic linkages, the structure is consistent in principle with that of GTF-SI, a dental caries pathogenesis factor, which mainly catalyzes α(1–3) glycosidic linkages.5 Accession numbers Coordinates and structure factors have been deposited in the PDB with accession codes 3AIE

Crystal Structure of Glucansucrase

(apo), 3AIC (complexed with acarbose) and 3AIB (complexed with maltose). Supplementary materials related to this article can be found online at .doi:10.1016/j.jmb.2011.02.028

185

10.

11.

Acknowledgements This study was supported in part by the Exploratory Research for Advanced Technology Iwata Human Receptor Crystallography Project (Japan Science and Technology Agency) (to S.I.), the Targeted Proteins Research Program (to S.I. and T.M.), Grants-in-Aid for Japan Society for the Promotion of Science Fellows (to K.I.), Grand-inAid for Young Scientists (B) (20780078) (S.I.), Grants-in-Aid for Scientific Research (B) (to T.K. and T.S.), a grant from the Research and Development Program for New Bioindustry Initiatives (to K.A.) and Grants-in-Aid for Scientific Research (S) (to K.A.) in Japan. The X-ray diffraction data collection was approved by the Photon Factory Advisory Committee (Proposal 2010G169).

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