From the
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,
¶ Harima Institute, The Institute of Physical and Chemical Research (RIKEN), 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 678-5148, Japan,
Department of Bioscience and Biotechnology, Aomori University, 2-3-1 Kohbata, Aomori 030-0943, Japan
Received for publication, December 23, 2002
, and in revised form, February 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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According to Henrissat's classification (8, 9), TLGT belongs to family 57 of the glycoside hydrolases. Most family 57 enzymes catalyze reactions similar to those of some -amylase family members (families 13, 70, and 77). However, no sequence similarity has been detected between family 57 and
-amylase family enzymes. The three-dimensional structures of many
-amylase family enzymes, including that of Thermus aquaticus amylomaltase (10), which produces large CAs, have been determined (11, 12, 13), and the amino acid residues involved in the catalysis have also been studied extensively (for a review, see Ref. 14). In contrast to
-amylase family enzymes, family 57 enzymes have received less investigation. Although the catalytic nucleophile of TLGT was recently determined (15), its three-dimensional structure, acid/base catalyst, and mechanism for large CA production remain unknown.
Previously, we made a preliminary report of the crystal structure of TLGT (16). In this paper, we describe the detailed structures of TLGT with and without a tetrasaccharide inhibitor, acarbose. The structures revealed the residues involved in catalysis and substrate binding of the enzyme and provided insight to investigate the mechanism for the production of large CAs.
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EXPERIMENTAL PROCEDURES |
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Data CollectionA multiple wavelength anomalous dispersion data set for a seleno-methionine-labeled form I crystal was collected at wavelengths of 1.0400 Å (remote), 0.9793 Å (peak), and 0.9795 Å (edge) at 100 K on beamline BL45PX of SPring-8 (Hyogo, Japan). A native data set for a form II crystal was collected at a wavelength of 1.0200 Å at 100 K on beamline BL45PX of SPring-8. An acarbose-bound data set for a form II crystal was collected at a wavelength of 1.0000 Å at 100 K on beamline BL18B of the Photon Factory (Tsukuba, Japan). The first two data sets were processed with DENZO and Scalepack (17), and the last one was processed with Mosflm (18).
Phase Calculation and RefinementFor phase calculation of the multiple wavelength anomalous dispersion data set, the program SOLVE (19) was used. The multiple wavelength anomalous dispersion map was improved by density modification using the program DM (20). The model for "form I-Se" was built into the resultant electron density map using the program O (21) and refined to 2.8 Å resolution, including simulated annealing, bulk solvent correction, and grouped B-factor refinement with the program CNS (22). Water molecules were picked automatically from Fo - Fc electron density maps using the program CNS and checked manually at a graphic station. The phase for form II-free crystal was calculated by molecular replacement using form I-Se as the initial model using the program CNS. Noncrystallographic symmetry was found on the self-rotation function, but it was not used for cross-rotation function nor translation function. Density modification was not applied to the molecular replacement solution. The calculated model was refined to 2.4 Å resolution, including rigid body minimization, simulated annealing, and grouped B-factor refinement with the program CNS. The two TLGT monomers in the asymmetric unit were not restrained during refinement, because there are some variations in the conformation of the two molecules as described later. The final model was found to exhibit good geometry, as determined using the program Procheck (23); 88.7% of the residues have /
angles in the "most favored region" of a Ramachandran plot. The model for form II complex was also refined using the program CNS. During refinement of form II complex, the ligands were added based on 2Fo - Fc and Fo - Fc electron density maps. The refinement statistics are presented in Table I. A structural data base search was performed using the DALI server (24). Least mean square fitting of the structures was carried out with the program LSQMAN (25). The oligosaccharide model shown in Fig. 7 was constructed using the program XtalView (26).
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Site-directed Mutagenesis and Enzyme AssayThe D214N mutant was generated with a QuikChange site-directed mutagenesis kit (Stratagene). An oligonucleotide with the sequence 5'-GTGTTCCATGACAATGGTGAAAAGTTCGG-3' and a complementary oligonucleotide, which replaced the codon for Asp214 (GAC) with AAT and introduced a HphI site for rapid screening of the mutation, were used. The mutation was reconfirmed by sequencing with an ABI PRISM 310 DNA sequencer (Applied Biosystems). Activity toward maltotriose was measured as described previously (27). One unit of activity was defined as the amount of enzyme that liberated 1 µmol of glucose from maltotriose per min at 80 °C.
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RESULTS |
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Domain IThe core of domain I is a (/
)7 barrel (residues 1258), in which seven central parallel
-strands (
1,
2,
3,
4,
5,
7, and
8) are flanked by helices (Fig. 3a). The (
/
)7 barrel is followed by a helical region that consists of 10 helices including the three-helix bundle made of
16,
17, and
18 (Figs. 1a and 3b) and covers the C termini of the (
/
)7 barrel, forming a cleft between them. The observation that acarbose was bound in the cleft in the form II complex (see below) and that the catalytic nucleophile Glu123 (15) was located in the middle of the cleft indicates that the cleft is an active site and domain I is a catalytic domain.
Domain IIDomain II of TLGT is composed entirely of -strands, with the exception of two short
-helices (
19 and
20). This domain is characterized by a
-sandwich fold, in which two layers of anti-parallel
-sheets are arranged in a nearly parallel manner. The layer adjacent to domain I consists of 10
-strands (
10,
11,
12,
13,
14,
15,
20,
21,
22, and
23), and the other layer consists of seven
-strands (
16,
17,
18,
19,
21,
22, and
24).
21 and
22 bend sharply and lie across the two layers. The same fold is found in chondroitin AC lyase from Flavobacterium heparinum (Protein Data Bank code 1CB8
[PDB]
),
-galactosidase from E. coli (1BGL
[PDB]
), hyaluronate lyase from Streptococcus pneumoniae (1EGU
[PDB]
), methylamine oxidase from Hansenula polymorpha (1A2V
[PDB]
), and copper amine oxidases from pea seedlings (1KSI
[PDB]
) and E. coli (1OAC
[PDB]
). Although the function of domain II is unclear at present, this domain may play a role in transglycosylation reaction of TLGT, because it has been suggested that
-sandwich domain of E. coli
-galactosidase is involved in transglycosylation reaction (28).
Calcium-binding SiteA calcium ion was bound at the loop between 10 and
11 in domain II (Figs. 1a and 2). The calcium ion was coordinated with O
1 of Asp392, O
2 of Asp394, O
1 of Asp396, the main chain carbonyl oxygen of Arg398, and O
1 and O
2 of Glu400.
In form II-free, one additional calcium ion was identified between Glu60 of chain B and Asn248 of chain A of an adjacent asymmetric unit (data not shown). Because no form II crystals were observed when calcium chloride was omitted from the crystallization buffer, the latter calcium ion may promote crystallization by tightening the interaction between the two TLGT molecules.
Catalytic ResiduesAn acarbose-TLGT complex was obtained by soaking a form II crystal in a buffer containing acarbose. The structure of the complex was determined at 2.4 Å resolution and refined to an R-factor of 19.8% (Table I). Electron density corresponding to an intact acarbose molecule was clearly observed for the active site of chain A (Fig. 4a). As expected, the acarbose molecule bound to subsites -1 to +3, where the acarviosine moiety, the inhibitory disaccharide group of acarbose, occupied subsites -1 and +1 and the maltose moiety occupied subsites +2 and +3 (Fig. 5a). The nomenclature for the subsites is according to Davies et al. (29). This is the inhibitory binding mode observed for most of the structures of -amylase family enzymes in complex with acarbose (30). Enzymatic hydrolysis of glycosidic linkages can be classified into two major types according to the anomeric configuration of the product, retaining and inverting, and in both cases the catalytic residues are typically two carboxylates (31). In retaining glycoside hydrolases, such as TLGT (2, 15), one residue acts as a nucleophile and the other as an acid/base catalyst. Glu123 is close (3.15 Å) to the C1 atom of the valienamine moiety at subsite -1, allowing nucleophilic attack (Fig. 4c), which is consistent with the results of a cross-linking study that demonstrated Glu123 to be a catalytic nucleophile (15). It is considered most likely that Asp214 is the acid/base catalyst of TLGT, because O
2 of Asp214 is only 2.96 Å away from the amide group of the valienamine moiety (this amide group is replaced by a glucosidic oxygen in a native substrate) (Fig. 4c), and there is no other acidic residue nearby. The average distances between all four pairs of O atoms of Glu123 and Asp214 (6.72 and 6.97 Å in the acarbose-free and acarbose complex structures, respectively) are in appropriate range for retaining enzymes (32). We generated the D214N mutant, in which Asp214 was replaced by Asn. The specific activity of the D214N mutant (0.0016 units/mg) was decreased about 10,000-fold as compared with that of the wild-type enzyme (17.7 units/mg). These results indicate that Asp214 is the acid/base catalyst.
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Subsite StructureFig. 5a shows interactions between acarbose and the protein. The valienamine moiety at subsite -1 is within hydrogen bonding distance of His11 and Asp354 and interacts with His13, Glu216, and Trp357 via water molecules. The 6-deoxyglucose moiety is fixed at subsite +1 with Arg124, Asp213, and Asp214 through hydrogen bonds, with Tyr272 through aromatic stacking, and with Trp221 through a hydrophobic interaction. The glucose moiety at subsite +2 is bound to Arg182 and Asp213 though hydrogen bonds, to Tyr183 through an aromatic stacking interaction, and to Trp221 through a hydrophobic interaction. The glucose moiety at subsite +3 seems to be more flexible, because it only exhibits a stacking interaction with Phe187 and a hydrophobic interaction with Trp221, i.e. no hydrogen bonding interaction with the protein.
In contrast to chain A, acarbose did not bind at the active site of chain B of the form II complex, and only a Tris molecule was identified there (data not shown). Tris was also found at the active site of chain B of the form II-free (Fig. 1a). Although acarbose did not bind to the active site, we found electron density corresponding to disaccharide, which seems to be maltose, at the edge of the active site cleft of chain B (Fig. 4b). The reducing end of maltose is 14.5 Å apart from subsite -1, suggesting that this binding site corresponds to subsites -5 and -6. The glucose moiety at subsite -5 is bound to His368 through a hydrogen bond and to Phe19 through a hydrophobic interaction (Fig. 5b). The glucose moiety at subsite -6 is within hydrogen bonding distance of Arg371 and exhibits hydrophobic interactions with Phe19 and Tyr601 (Fig. 5b). In chain A, maltose did not bind to this site, because this region was involved in the crystal contact. From the complex structure with acarbose, TLGT was revealed to possess at least nine subsites, -6 to +3, although the residues forming subsites -4 to -2 could not be determined in this study.
Unexpectedly, a glucose molecule was found at subsite +1 of form I-Se (data not shown). Glucose interacted with Arg124, Asp214, Asp213, and Tyr272. Interactions between glucose and these residues in form I-Se are the same as those observed between the +1 glucose moiety of acarbose and the corresponding residues in the form II complex. Glucose was probably derived from trehalose reagent, which was used as a cryoprotectant and contained a trace amount of glucose.
The active site cleft of TLGT is tunnel-like in shape, as evidenced by the three lids that cover the cleft (Figs. 6 and 7). The first lid (lid 1, residues 220224) protrudes from the (/
)7 barrel (Fig. 2). The second (lid 2, residues 358363) and third (lid 3, 627630) lids protrude from the three-helix bundle and domain II, respectively (Fig. 2). Upon binding of acarbose, the conformations of lids 2 and 3 change significantly (Fig. 6). In the absence of acarbose, the side chains of Val360 and Phe361 are directed toward the active site cleft. The bound acarbose collides with the side chains of Val360 and Phe361, leading to movement of lid 2. This movement induces a large movement of lid 3 (Fig. 6). For example, the C
of Ser627 and Glu628 move 4.2 and 6.2 Å, respectively. In addition to the movements of these two regions, the
1 axis of Tyr183 and the
2 axis of Phe187 rotated to interact with the pyranose rings of the maltose moiety via hydrophobic stacking at subsites +2 and +3, respectively (Fig. 6).
Subunit InterfaceAlthough the TLGT gene encodes a 78-kDa polypeptide, purified TLGT is eluted as a 168-kDa protein in gel filtration chromatography using Superdex 200 column (Amersham Biosciences) (data not shown). This indicates that TLGT is a homodimer, as previously suggested by Xavier et al. (1). In a form II crystal, two TLGT monomers, which are in an asymmetric unit, interact via the same surface of domain I, because there is a pseudo-2-fold axis between them (Fig. 1a). In a form I crystal, two TLGT monomers, which are in adjacent asymmetric units associated through a 2-fold axis, interact in the same manner (data not shown). This observation indicates that the two TLGT monomers interact in the same manner as observed in these crystals. Two hydrogen bond networks and a hydrophobic patch form the primary contribution to the interactions between the two subunits (Fig. 8a). One hydrogen bond network is constructed from two water molecules and six residues: Glu166 and Tyr266 from one monomer, and Ala91, Lys314, Asn324, and Lys328 from the other (Fig. 8a). The hydrophobic patch is formed by Leu287, Phe288, Phe291, Leu295, Tyr304, Phe307, and Val308 (Fig. 8b). In particular, Leu295 and Val308 are conserved (Fig. 2) and in contact with their counterparts in the other monomer (Fig. 8b), which suggests that they play a central role in the hydrophobic contact in this region. The proportion of the buried surface area (1500 Å2) is large enough for dimerization, compared with the total molecular surface of a monomer (
22300 Å2). Oligomerization is known to be one of the strategies by which proteins acquire thermostability (33). Because the dimer interface is located on the opposite side of the active site, dimerization seems to contribute to the thermostability rather than the activity including amylose cyclization.
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DISCUSSION |
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Previously, we found that the nucleophilic residues of TLGT and class II -mannosidases (glycoside hydrolase family 38) are located at the same position in the amino acid sequences despite their low sequence similarity (15). When the two families were compared at the three-dimensional level, striking structural similarities were observed in the catalytic domains of TLGT (Fig. 3b) and Drosophila melanogaster Golgi
-mannosidase II (37) (Fig. 3c): a
-sheet core surrounded by
-helices, a three-helix bundle, and a catalytic nucleophile at the fourth
-
loop. These findings strongly suggest that the two families have evolved from a common ancestor. Despite the similarities, however, there are also significant structural differences between the two enzymes. The most important one is that the catalytic domain of
-mannosidase II does not have a barrel structure, because there is no hydrogen bond that connects the two
-strands (7 and 8 in Fig. 3c) corresponding to
7 and
8 of TLGT. The
fold in
-mannosidase II may be a result of protein evolution from the (
/
)7 barrel fold found in TLGT and vice versa. This difference is interesting from the standpoint of the evolution of protein folding.
In the crystal structures of amylase-acarbose complexes, acarbose was often found in a modified form, because of the transglycosylation activity of amylase (see Ref. 30). However, it is clear that acarbose bound to the active site of TLGT (chain A in the form II-complex) is an intact one, because the electron density indicates that the sugar ring at the subsite -1 is somewhat flattened and that O-6 is not present at the subsite +1. Why maltose bound to the subsite -5 and -6 of chain B in the form II-complex is uncertain. Because maltose was not detected in the acarbose reagent when analyzed on thin layer chromatography (data not shown), acarviosine moiety of acarbose may have flexible conformation, and only maltose moiety may be observed as a clear electron density. Additional electron density at the nonreducing end seen in Fig. 4b can be explained by this idea. Such a case has been also reported in the crystal structure of Bacillus circulans xylanase complexed with xylotetraose, in which electron density for only two xylose residues was observed (38). It is also unclear why acarbose did not bind to the active site of chain B in the form II crystal. Although TLGT has a larger Ki value (0.6 x 10-3 M) for acarbose than
-amylase family enzymes (0.6 x 10-7 to 0.8 x 10-4 M) (39), the acarbose concentration (10 mM) in the soaking solution is sufficient. One possibility is that conformational changes in chain B prevent the binding of acarbose. When compared with form I and chain A of form II, the slight movements of
8,
9,
10,
11,
13,
14, 3105,
22, and
23, and the destruction of some ion pairs in chain B were observed (data not shown), probably because of crystal packing. These movements seem to change chain B into an inactive form that cannot bind the substrate.
There are several kinds of 4--glucanotransferases, including cyclodextrin glucanotransferase (CGTase), many of which produce CAs (6). The minimum ring size of CAs varies with the enzyme. The smallest products of CGTase are CAs with 68 glucose units. Potato 4-
-glucanotransferase (3) and T. aquaticus amylomaltase (40) produce CAs with 17 or more and 22 or more glucose units, respectively. Sixteen is the minimum ring size of CAs produced by TLGT2 and Thermococcus kodakaraensis 4-
-glucanotransferase (also a family 57 enzyme) (41). What determines the minimum ring size of CAs produced by these enzymes? It has been proposed that in the case of large CA formation by T. aquaticus amylomaltase, the product wraps around the bulky loop, which partially covers the active site (30). In addition to the loop structure, it was thought that the existence of a second ligand-binding site apart from the catalytic site also contributed to the large CA formation (30). In contrast, there is no such large steric hindrance in CGTase. Although T. aquaticus amylomaltase and TLGT have different structures, they seem to have adapted a similar strategy to produce large CAs. In the form II-complex of TLGT, acarbose partially wraps around lid 1 (Fig. 7). Lid 1 seems to prevent the formation of small CAs because of its considerable steric hindrance. Because amylose forms a helical structure in which glucose units are connected through O-3O-2' hydrogen bonds,
-1,4-linked glucose polymers tend to become circularized in an aqueous solution. In fact, the reorganization of O-3O-2' hydrogen bonds is proposed to be one of the forces in the circularization step responsible for the formation of cyclodextrins by CGTase (42). If there is no steric hindrance by the loop observed in TLGT and amylomaltase, smaller CAs might be produced. When we included a polysaccharide chain in the structure of chain A of the form II complex, a chain of 14 glucose residues was built into the structure, in addition to the tetrasaccharide inhibitor at the catalytic site (Fig. 7). This polysaccharide model and acarbose correspond to a CA with 18 glucose units. Because TLGT predominantly produces a CA with 1820 glucose units,2 this model seems to represent the most common mode of CA binding in TLGT. When the minimum ring size of CA (16 glucose residues) is produced, some assumption need to be made. From this model, in addition to lid 1, the large distance (
27.5 Å) between subsites -6 and +3, which is caused by the relatively extended structure of the cleft, coupled with the steric hindrance of the side chains of Phe19 and Trp21 also seem to prevent the formation of small CAs in TLGT (Fig. 7). When amylose circularizes, the nonreducing end of a polysaccharide chain must travel a considerable distance to the acceptor site in the active site. It has been proposed that the movement of some hydrophobic residues assists in the circularization of a substrate in CGTase (43). The structural movements observed in TLGT (Fig. 6) may also facilitate the circularization of amylose.
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FOOTNOTES |
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* This work was supported in part by Grants-in-Aid for Scientific Research 10460035 and 12460047 (to H. M.) from the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship for young researcher of the Japan Society for the Promotion of Science. To whom correspondence may be addressed. Present address: ATP System Project, ERATO, JST, 5800-3 Nagatsuta, Midori-ku, Yokohama 226-0026, Japan. Fax: 81-45-92-5239; E-mail: himamura-ra{at}res.titech.ac.jp.
|| Present address: Dept. of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
** Present address: Dept. of Microbiology, College of Medicine, Gyeongsang National University, 90 Chilam-Dong, Jinju, Gyeong-Nam 660-750, Korea.
To whom correspondence may be addressed. Fax: 81-17-738-2030; E-mail: hmatsuza{at}aomori-u.ac.jp.
1 The abbreviations used are: TLGT, 4--glucanotransferase from T. litoralis; CA(s), cycloamylose(s); CGTase, cyclodextrin glucanotransferase.
2 B. S. Jeon and H. Matsuzawa, unpublished results.
3 H. Imamura and H. Matsuzawa, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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