The crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II (TVA II) complexed with transglycosylated product Masahiro Mizuno 1 , Takashi Tonozuka 1 , Akiko Uechi 1 , Akashi Ohtaki 2 , Kazuhiro Ichikawa 1 , Shigehiro Kamitori 2 , Atsushi Nishikawa 1 and Yoshiyuki Sakano 1 1 Departments of Applied Biological Science and 2 Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan An a-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes a-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes a-1,4-glucosidic linkages of cyclodextrins and a-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4 3 -a-panosylpanose (4 3 -P2) (a pullulan hydro- lysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4 2 -a-pano- sylpanose (4 2 -P2), which was produced by transglycosyla- tion from 4 3 -P2, at 2.2-A ˚ resolution. The shape of the active cleft of TVA II is unique among those of a-amylase family enzymes due to a loop (residues 193–218) that is located at the end of the cleft around the nonreducing region and forms a ÔdamÕ-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-li- ganded state and the other is supplying the water that is necessary for substrate hydrolysis. Keywords: a-amylase; GH family 13; 4 2 -a-panosylpanose; substrate specificity; transglycosylation. a-Amylase (1,4-a- D -glucan-4-glucanohydrolase; EC 3.2.1.1) hydrolyzes a-1,4-glucosidic linkages of starch to release a-anomer products. Numerous enzymatic properties of a-amylase have been reported, due to the industrial importance of this enzyme in food and pharmaceutical fields. According to the classification system proposed by Henrissat et al. [1–3], a-amylases are classified into glycoside hydrolase (GH) family 13. Thermoactinomyces vulgaris R-47 produces a-amylase II (TVA II) as an intracellular enzyme [4]. TVA II hydrolyzes a-1,4-glucosidic linkages of starch like other a-amylase family enzymes to produce mainly maltose. In addition to a-amylase activity, TVA II hydrolyzes a-1,4-glucosidic linkages of pullulan to produce panose [5,6] via an activity proposed as neopullulanase activity by Kuriki et al.[7]. TVA II also hydrolyzes a-1,4-glucosidic linkages of cyclo- dextrins [8] and a-1,6-glucosidic linkages of isopanose [9,10]. The crystal structure of TVA II has been determined at 2.3-A ˚ resolution [11,12], and TVA II has been shown to form a dimeric structure (Fig. 1A). Each monomeric subunit of TVA II is composed of four structural domains, N (residues 1–121), A (residues 122–242 and 298–502), B (residues 243–297), and C (residues 503–585) (Fig. 1B). Domain A forms a (b/a) 8 -barrel structure that is the catalytic unit containing three catalytic residues (Asp325, Glu354 and Asp421), which is typical of a-amylase family enzymes. Domain B is a small component which protrudes from the third b-strand of domain A. Domain C is also highly conserved among a-amylase family enzymes, but its function is still not so clear. TVA II has a notable extra domain consisting of 120 amino acid residues at the N-terminus, called domain N, which appears to be involved in forming the dimeric structure [13]. The N domains of both molecules are involved in forming each of the active clefts in cooperation with the A domains. TVA II shows broader substrate specificity than other a-amylase family enzymes: for example, it hydrolyzes a-1,4- glucosidic linkages of starch, pullulan and cyclodextrin, and a-1,6-glucosidic linkages of isopanose. It is still unclear what accounts for this broad substrate specificity of TVA II. We have already reported the structures of TVA II complexed with maltotetraose [14] and cyclodextrins [14,15], while the structure of the complex with an oligosaccharide based on pullulan has not been analyzed. In this study, to analyze the pullulan recognition mechanism, we first developed a Correspondence to Y. Sakano, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-Cho, Fuchu, Tokyo 183-8509, Japan. Fax: + 81 42 3675705, Tel.: + 81 42 3675704, E-mail: sakano@cc.tuat.ac.jp Abbreviations: TVA II, Thermoactinomyces vulgaris R-47 a-amylase II; GH, glycoside hydrolase; PEG, polyethylene glycol; MPD, 2-methyl-2,4-pentanediol; 4 3 -a-panosylpanose (4 3 -P2), Glcp(a1 fi 6)Glcp(a1 fi 4)Glcp(a1 fi 4)Glcp(a1 fi 6) Glcp(a1 fi 4)Glc; 4 2 -a-panosylpanose (4 2 -P2), Glcp(a1 fi 6) Glcp(a1 fi 4)Glcp(a1 fi 4)[Glcp(a1 fi 6)] Glcp(a1 fi 4)Glc) (Glcp(a1 fi 6)Glcp(a1 fi 4)Glcp(a1 fi 4)[Glcp(a1 fi 6)] Glcp(a1 fi 4)Glc. Enzyme:1,4-a- D -glucan-4-glucanohydrolase (EC 3.2.1.1). (Received 25 February 2004, accepted 23 April 2004) Eur. J. Biochem. 271, 2530–2538 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04183.x method to form a complex between TVA II and a pullulan model substrate by using partial hydrolyates of pullulan and an inactive TVA II mutant, D325N. A hexasaccharide containing two panose units, 4 3 -a-panosylpanose (4 3 -P2) (Fig. 2A), was thereby prepared. We analyzed the crystal structure of the complexed form at 2.2-A ˚ resolution and found that the transglycosylation product was bound in the active cleft. Materials and methods Gene construction for Y374A-TVA II mutant The gene manipulation methods were based on those of Sambrook et al. [16]. Site-directed mutagenesis was carried out using plasmid pTN302-10 as described [8] according to the method of Kunkel et al. [17]. To construct the Y374A mutant, the following oligonucleotide was used as a mutagenic primer: 5¢-GATCACACTCTCGCG AAACA AATAATTCATCACCG-3¢. The underlined nucleotide in the primer indicates the mismatched nucleotide creating the alanine substitution mutation. DNA sequencing confirmed the presence of the mutation. The gene construction for the D325N mutant has already been reported [18]. Purification, crystallization and data collection The mutated TVA II was prepared using recombinant Escherichia coli MV1184 cells and was purified as described [19]. The crystals of D325N were grown at 20 °C using the hanging-drop method, in which 1.5 lLof a20mgÆmL )1 D325N solution in 5 m M Tris/HCl buffer (pH 7.5) was mixed with the same volume of a reservoir solution containing 1% (w/v) PEG6000, 5 m M CaCl 2 in 40 m M Mes/NaOH (pH 6.1). The crystal complex of D325N with 4 3 -P2 prepared by the same method as described in our previous paper [20] was obtained by soaking the crystal in cryo-protectant solution [20% (w/v) PEG6000, 20% (v/v) MPD, 2.5 m M CaCl 2 ] containing 10 m M 4 3 -P2 for 10 h. The diffraction data was collected at the beam line of BL18B, PF (Photon Factory, Japan). The data was processed and scaled using the programs DPS / MOSFLM [21]. Structure refinement The structure of the D325N complex was solved by molecular replacement using the unliganded TVA II as thesearchmodel.The2F o –F c electron density map showed that a continuous density 1 r contoured level for all atoms of the protein is seen except for Ser276- Arg280 of both subunits. After simulated annealing refinement using the program CNS [22], the different Fourier maps clearly revealed a density corresponding to a hexasaccharide. Water molecules were added automat- ically using CNS and a 3.0 r cut-off for peaks in F o –F c maps. To avoid overfitting of the diffraction data, a free R factor with 10% of the test set excluded from Fig. 1. Ribbon representation of the fold of TVA II in complex with 4 2 -P2. (A) Dimeric form. (B) Monomeric form. MOL-1, MOL-2 and each domain are shown by different gray scales. Darker hues are used for MOL-1. Names of each domain with or without the asterisk represent MOL-1 or MOL-2. Three catalytic residues are drawn in black stick and 4 2 -P2 molecules are drawn in red sticks. The bound calcium ions are shown as black spheres. Figures were produced with MOLSCRIPT [35] and RENDER from the R ASTER 3D package [36]. Ó FEBS 2004 Structure of T. vulgaris a-amylase II complex (Eur. J. Biochem. 271) 2531 refinement was monitored [23]. Refinement of the final structure were converged at an R factor of 0.194 (R free ¼ 0.233), and contained 1170 amino acid residues, two cal- cium ions, two 4 2 -P2 molecules and 399 water molecules. Model quality and refinement statistics Refinement statistics are presented in Table 1. Analysis of the Ramachandran plot [24], calculated with the program PROCHECK [25], revealed that 86.2% of residues in MOL-1 and 84.9% of residues of MOL-2 were in the most favored region, and only one residue (Thr278 of MOL-2) was found in disallowed region. Protein Data Bank accession number The atomic coordinates and structure factors of the D325N complex (PDB code 1VB9) have been deposited in the Protein Data Bank. Kinetic study Purified enzyme (diluted to 0.01 mgÆmL )1 ,120lL) was addedto480 lL of various concentrated substrates (soluble starch was purchased from Merck, Germany; pullulan was obtained from Hayashibara Biochemical Laboratories, Japan) in 100 m M sodium phosphate buffer (pH 6.0), and the hydrolysis reaction was started at 40 °C, with sampling every 5 min. After the reaction had stopped, the method of Somogyi-Nelson [26] was followed. Table 1. Data collection and refinement statistics. 4 2 -P2 complex Data collection Temperature (K) 100 Space group P2 1 2 1 2 1 Cell dimensions a(A ˚ ) 113.1 b(A ˚ ) 118.3 c(A ˚ ) 112.1 a ¼ b ¼ c (°)90 Resolution range (A ˚ ) 34–2.2 Number of measured references 378751 Number of unique references 76785 Completeness (%) 99.8 (99.8) b R merge a 0.053 (0.236) b I/r(I) 10.6 Structure refinement Resolution range (A ˚ ) 34.0–2.2 Numbers of references 76732 R 0.194 (0.207) b R free 0.233 (0.249) b Completeness (%) 99.8 (99.7) b rmsd bond lengths (A ˚ ) 0.006 rmsd bond angles (°) 1.3 Number of amino acids 1170 Number of solvent molecules 508 a R merge ¼ SS|I i –<I>|/S<I>. b The values for the highest resolution shell are given in parentheses (2.34–2.20-A ˚ resolution). Fig. 2. Topologies of pullulan model oligosaccharides. (A) 4 3 -a-Panosylpanose is abbreviated 4 3 -P2. (B) 4 2 -a-Panosylpanose is abbreviated 4 2 -P2. (C) The F o –F c electron density map of 4 2 -P2 bound at the active site. The number of glucose units is labeled from )3 (nonreducing end) to +2 (reducing end), except for +2¢, which branches from +1 with an a-1,6-glucosidic linkage in 4 2 -P2. The contour level is 2.0 r. 2532 M. Mizuno et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Results Carbohydrate in the catalytic site The structure of the complex was determined by molecular replacement using the structure of unliganded TVA II (PDB code 1JI2) [12] as a search model. In the final model, there were two subunits (MOL-1 and MOL-2) related by noncrystallographic twofold sym- metry, in an asymmetric unit (Fig. 1A). MOL-1 and MOL-2 are homodimers. The overall structure of the complex form was essentially identical to that of unliganded TVA II except for the induced fitting of some regions, residues 137–170, residues 417–425 and residues 455–481, composed the catalytic cleft. The root mean square deviation value calculated for the whole Ca chain is 1.10 A ˚ . After the initial structural refinement, the difference Fourier map indicated that the clear continuous electron densities for the pentasaccharide consisted of the five glucose units, labeled Glc )3, )2, )1, +1 and +2, at the active sites of both MOL-1 and MOL-2. This pentasaccha- ride occupied subsites )3 to +2 (The subsites are numbered based on the nomenclature of Davies et al. [27]). Also, weak density was seen around the O6 of Glc +1 at the center of the active site. After refinement with this pentasaccharide, the rest of the density was clearly shown. This new density was assigned as a glucose unit and labeled as Glc +2¢,and its electron density between O1 of Glc +2¢ and O6 of Glc +1 seemed to be connected with the a-1,6-glucosidic linkage (Fig. 2C). Therefore, the oligosaccharide bound at the active site was determined to be 4 2 -a-panosylpanose (4 2 -P2) (Fig. 2B). The most surprising thing about these results is that the mutated enzyme was soaked in a solution of 4 3 -P2 (Fig.2A),butthat4 2 -P2 (Fig. 2B) was actually bound in theactivesiteinsteadof4 3 -P2. In TVA II, Asp325, Glu354 and Asp421 have been identified as the catalytic residues [18]. The activity for pullulan of the D325N used in the crystallization was less than 0.006% that of the wild-type enzyme [18]. However, D325N released a small amount of panose from pullulan at a high enzyme concentration with a long reaction time, as observed by thin layer chromato- graphy [18]. TVA II also carries out a transglycosylation reaction to form both a-1,4- and a-1,6-glucosidic linkages [20]. Thus, it is possible that 4 2 -P2 is produced by a transglycosylation reaction. 4 2 -P2 binding The complexed structure enables a detailed analysis of the interactions of the active site with 4 2 -P2. To facilitate description of these interactions, the active cleft of TVA II is separated into two parts called the nonreducing region (containing subsites )1, )2and)3) and the reducing region (containing subsites +1, +2 and +2¢)in this report. TVA II forms a homodimeric structure, while most of the a-amylases form a monomeric structure. Although the active cleft of TVA II in the monomeric structure is wide and shallow, domain N of MOL-2 contributes to the formation of a narrow, deep cleft around the reducing region in the dimeric structure, while the nonreducing region is not affected by formation of the dimeric structure (Fig. 3A). Yokota et al.[13]constructeda mutated TVA II truncated domain N and showed that domain N was necessary for the formation of the dimeric structure and enzymatic activities. The average tempera- ture factors for Glc )3, )2, )1, +1 and +2 are 36.1, 27.6, 28.8, 35.1 and 42.2 A ˚ 2 , respectively. The values for Glc )1and)2 are lower than those for the other glucose units because the maltose unit bound at subsites )1and )2 is taken up into the bottom of the active cleft and tightly bound to the enzyme by multiple hydrogen bonds. Table 2 lists the hydrogen bond environments of the bound 4 2 -P2. Non-reducing region Figure 3(B) shows the residues engaged in 4 2 -P2 binding at the nonreducing region. O2 and O3 of Glc )1form hydrogen bonds with Asp421 at distances of 2.4 and 2.7 A ˚ , respectively. Asp421 is one of the three catalytic residues and the pullulan-hydrolyzing activity of mutated TVA II (D421N) was drastically decreased to 0.001% of that of the wild-type enzyme [18]. Asp421, which has been proposed to function as a ÔfixerÕ for Glc )1, causes deformation of the glucose ring, which is essential for the catalysis [28]. In this complexed structure, the conformation of the ring of Glc )1 was slightly distorted from the 4 C 1 chair form. Asp465 and Arg469 are involved in the binding of Glc )2. Asp465 interacts with O3 of Glc )2 at a distance of 2.6 A ˚ and Arg469 also interacts with O2 and O3 of Glc )2 at distances of 2.8 and 3.1 A ˚ , respectively. The recognition of the maltose unit by Asp421, Asp465 and Arg469 is widely found in a-amylase family enzymes, indicating that this mode of maltose recognition is a common mechanism regardless of the diversity of the substrate specificity. His202 is located at the bottom of the active cleft, and only forms a hydrogen bond with the O2 of Glc )3 at the distance of 2.8 A ˚ . Reducing region While many interactions between the enzyme and the substrate were identified in the nonreducing region, relat- ively few interactions with the substrate were seen in the reducing region (Fig. 3C). Remarkable conformational changes of two amino acid residues around subsite +2, Trp356 and Tyr374, were observed between the unliganded and complexed structure. Once 4 2 -P2 is taken into the active site, the side chain of the Trp356 is rotated from )174.7° to 169.4° in torsion angle of C c –C b –C a –C on Trp356. This conformational change makes a plane of its side chain parallel to the ring of Glc +2 and contributes to interaction with Glc +2 through a stacking effect. Furthermore, this adjustment of Trp356 seems to trigger a rotational change of Tyr374. The side chain of Tyr374 is rotated from )51.1° to 149.8° in torsion angle of C c –C b –C a –C on Tyr374 without the steric barrier of Trp356, and also precisely becomes parallel to Trp356 and Glc +2. The reducing region of the active cleft is coordinately composed of domain A of MOL-1 and domain N of MOL-2. Two loops (Asp43-Glu51 and Glu104-Tyr113) of Ó FEBS 2004 Structure of T. vulgaris a-amylase II complex (Eur. J. Biochem. 271) 2533 domain N appear to be strongly involved in substrate binding. O2 and O3 of Glc +2 form hydrogen bonds with Gln112 and Arg44, both of which belong to domain N of MOL-2, at distances of 2.9 and 3.3 A ˚ , respectively. Glc +2¢ occupied the center of the active site without any interactions with MOL-1 or MOL-2, and its average temperature factor was 48.9 A ˚ 2 . The configuration of the a-1,6-glucosidic linkage between Glc +1 and +2¢ was completely different from that between Glc )3and)2. The torsion angles of O6–C6–C5–C4 in Glc +1 and +2¢,and Glc )3and)2, were )168.8° and 47.2°, respectively. The distances between O2 (+2¢) and O6 (+2), and O2 ()3) and O6 ()1) were 4.2 and 6.7 A ˚ , respectively. Phe286 of MOL-1 and Tyr45 of MOL-2, which play important roles in the binding of cyclodextrins [29], are located at the nearest distances of 3.8 A ˚ and 4.1 A ˚ , and interact with Glc +2¢ via van der Waals force. In the structure of neopullulanase complexed with maltotetraose, the electron density corres- ponding to maltose was observed proximal to the position of Glc +2¢, and it was proposed that maltose may be a potential acceptor in the transglycosylation reaction [30]. Thus, these findings suggest that the position around Glc +2¢ has the ability to hold a monosaccharide or small oligosaccharide. A summary of the intermole- cular hydrogen-bonding interactions that can be inferred for the complex between TVA II and 4 2 -P2 is presented in Fig. 4. Discussion Four loops in the nonreducing region The nonreducing region of the active cleft, containing subsites )1, )2and)3, consists of four loops, loop I (residues 136–171), loop II (residues 193–218), loop III (residues 257–302), and loop IV (residues 454–482) (Fig. 5A). Loops III and IV are located at each side of the cleft to form a cleft, and the width of this cleft is about 10 A ˚ Fig. 3. Stereo-view of the active site with 4 2 -P2. (A) The whole shape of the active cleft formed collaboratively with domain N of MOL-2 (green surface model) is shown in the molecular surface model. The surface model was produced using PYMOL (http://www. pymol.org). (B) Unliganded TVA II (green) superimposed into the complex structure (magenta) around the nonreducing region. 4 2 -P2, separated between )1and+1,isdis- played as dark gray sticks. The residues with asterisks are located in domain N of the MOL-2 molecule. (C) Reducing region. The explanation is the same as for (B). 2534 M. Mizuno et al. (Eur. J. Biochem. 271) Ó FEBS 2004 at its narrowest. Loop I is also a component of the active cleft, but loop I does not directly interact with 4 2 -P2. Loop II is located in the end of the cleft composed of loops III and IV, and seems to act as a ÔdamÕ of the cleft. These four loops of TVA II are superimposed on those of a-amylase (Taka-amylase A) from Aspergillus oryzae (PDB code, 7TAA) [31] and cyclodextrin glucanotransferase (CGTase) from Bacillus circulans strain 251 (PDB code, 1CDG) [32] (Fig. 5A). The Ca backbones of loop IV, where several highly conserved residues, such as Asp465 and Arg469 of TVA II, are also located, are similar in these three enzymes. It appears that the shape of loop IV is necessary for the recognition of the maltose unit bound Glc )1and)2ina-amylase family enzymes. In contrast, loop III of TVA II adopts a different conformation from that of other a-amylase family enzymes. In TVA II, this loop is shorter than in these other enzymes, but the C-terminus of the loop is connected with domain B. Loop II is located at the end of the active cleft and forms a ÔdamÕ-like bank. In TAA and CGTase, loop II is 10 and 14 residues longer than that in TVA II, and protrudes more markedly into the active cleft compared to Loop II in TVA II (Fig. 5B). In most a-amylases, loop II makes a large bank in the active cleft, as in CGTase and TAA. In contrast, loop II of TVA II is short and the bank is small, which allows an open cleft. This distinctive shape of the cleft of TVA II enables TVA II to incorporate various substrates, including pullulan, into theactivecleft. We previously analyzed the structure of TVA II com- plexed with maltohexaose, but found that Glc )3was disordered [14]. We estimated the position of Glc )3ofthe a-1,4-glucan using the structure of TAA complexed with acarbose (Fig. 5C). The positions of the maltose unit, Glc )1and)2, are almost the same in the two enzymes. However, the position of Glc )3of4 2 -P2 is completely different from that of acarbose. In the case of 4 2 -P2, Glc )3 extends toward the space between loops II and III. The length of loop II of TVA II is very short and the cleft is open, which enables TVA II to bind pullulan efficiently. Loop II of TAA occupies the end of the active cleft round the nonreducing region, which seems to restrain the uptake of pullulan into the active site. Tyr75, located at Loop II of TAA, also seems to be a steric barrier to the uptake of Glc )3 in TAA. On the other hand, in acarbose, Glc )3 extends toward the space between loops I and II. Although Glu35, located at loop I of TAA, is engaged in the binding of Glc )3, His164 of TVA II, located at the position corresponding to Tyr75 of TAA, is too close to Glc )3 in this model. The activity of TVA II for starch and its derivatives is almost equal to that for pullulan. Thus, the hydrolysis of pullulan by TVA II appears to be the result of effective binding due to the shape of the active cleft around the nonreducing region. The substrate recognition of TVA II at the nonreducing region of the active cleft is different from those of other a-amylase family enzymes. These differences are also due to the individual amino acid residues that interact directly with substrate, but are mainly due to the shape of the active cleft composed of the four loops. Roles of Trp356 and Tyr374 Drastic conformational changes of two residues, Trp356 and Tyr374, were observed upon binding with 4 2 -P2 (Fig. 3C). In neopullulanase [30] and maltogenic amylase [33], the residues corresponding to Trp356 and Tyr374 are already stacked in the unliganded state. The 2F o –F c electron density maps of Trp356 and Tyr374 in unliganded and Table 2. Hydrogen bond contacts between TVA II and 4 2 -P2. Glucose number Glc. Atom TVA II atom Distance (A ˚ ) Glc )3 O2 His202-NE2 2.8 Glc )2 O2 Arg469-NH2 2.8 O3 Asp465-OD2 2.6 O3 Arg469-NH2 3.2 O3 Arg469-NH1 3.1 Glc )1 O2 Asp421-OD2 2.4 O2 His420-NE2 3.1 O2 Glu354-OE1 3.4 O3 Asp421-OD1 2.7 O3 His420-NE2 3.1 Glc +1 O6 Met293-SD 3.2 Glc +2 O3 Glu354-OE1 2.8 O2 Gln112 a -NE2 2.9 O3 Arg44 a -NH2 3.3 a Gln112 and Arg44 are located at domain N of MOL-2. Fig. 4. Schematic drawing of the interactions of 4 2 -P2 bound to the active site. Hydrogen bonds of less than 3.5 A ˚ are shown as dashed lines.Watermoleculesareshownasspheres. The residues with asterisks are located in domain N of the MOL-2 molecule. Three catalytic residues, except for Asn325, which is aspartic acid in native TVA II, are surrounded by an elliptical box. Ó FEBS 2004 Structure of T. vulgaris a-amylase II complex (Eur. J. Biochem. 271) 2535 Fig. 5. Four loops composing the nonreducing region of the active cleft. Stereo-views of the four loops that form the active cleft of TVA II (in magenta), which are superimposed on CGTase (PDB code, 1CDG) and TAA (PDB code, 7TTA), drawn with coils in orange and green, respectively. Loops I, II, III and IV are located at the nonreducing region of the active cleft. (A) The four loops and the residues engaged in substrate binding are shown in a coil and stick model. (B) The comparison of the shape of the active cleft based on the molecular surface model of TVA II. CGTase and TAA are superimposed and drawn in coils. (C) The position of Glc )3ofa-1,4-glucan is predicted using the structure of TAA complexed with acarbose. The nonreducing region of 4 2 -P2 (black stick) and acarbose (antique white sticks) are only shown as sites )1, )2and)3 for 4 2 -P2 and )1¢, )2¢ and )3¢ for acarbose. 2536 M. Mizuno et al. (Eur. J. Biochem. 271) Ó FEBS 2004 complexed TVA II were clearly seen (Fig. 6). When no substrates are taken into the active site, Tyr374 is fixed by Glu98 of MOL-2 with a weak hydrogen bond at a distance of 3.4 A ˚ , and the space around Trp356 is observed as a wide cavity. This environment generates the flexibility of Trp356, allowing suitable interaction with Glc +2. Tyr374 is continuously rotated to cause the stacking with Trp356, and thus Tyr374 seems to play an important role as lining for Trp356. To investigate the role of Tyr374, we constructed Y374A mutated TVA II, with replacement of tyrosine by alanine, by site-directed mutagenesis, and carried out kinetic analysis for starch and pullulan. The K m value of Y374A for starch was almost identical to that of the wild-type enzyme and that of Y374A for pullulan showed nearly a threefold decrease compared to that of the wild type. Because the cavity around Trp356 of the Y374A mutant was very wide in both the unliganded and liganded states, it is likely that in the mutant, Trp356 was enabled to rotate its side chain to be appropriate for the position of Glc +2. On the other hand, the k cat value of the Y374A mutant protein was decreased to less than 10% of the wild-type value (Table 3). This observation suggests that Tyr374 also participates in the catalytic activity, in addition to assisting in substrate binding through the lining of Trp356. Upon hydrolysis, a water molecule, located near the glucosidic linkages between Glc )1 and +1, is incorporated into the carbonium cation intermediate. Tyr374 in the substrate binding state catches a water molecule at a distance of 2.6 A ˚ ,andthiswaterisalso captured by two catalytic residues, Glu354 and Asp421, at thesamedistanceof2.7A ˚ . Kuriki et al. [34] suggested that Tyr377, Met375 and Ser42 of neopullulanase (correspond- ing to Tyr374, Met372 and Ser419 of TVA II) are located on the entrance path of the attacking water molecule, and these residues are involved in hydrolysis and transglycosy- lation, as shown by using site-directed mutagenesis and computer modeling. The replacement of tyrosine by alanine increases the hydrophobicity around the entrance path of the water molecule and makes it impossible to fix the water molecule near the glucosidic linkage to be cleaved. Thus, we suggest that Tyr374 is involved in supplying the water that is necessary for substrate hydrolysis. Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research (14580621) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The data collection was carried out under the approval of the Photon Factory Advisory Committee, the National Laboratory for High Energy Physics, Tsukuba (2001G341). We thank Dr Igarashi and Dr Suzuki for help in data collection at the Photon Factory, BL18B. We also thank the X-ray crystallography laboratory, Tokyo University of Agriculture and Technology, Fuchu, Tokyo for data collection using an R-AXISIIc. References 1. Henrissat, B. (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280, 309–316. 2. Henrissat, B. & Bairoch, A. (1993) New families in the classifica- tion of glycosyl hydrolases based on amino acid sequence simila- rities. Biochem. J. 293, 781–788. 3. Henrissat, B. & Bairoch, A. (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695–696. 4. Tonozuka, T., Ohtsuka, M., Mogi, S., Sakai, H., Ohta, T. & Sakano, Y. (1993) A neopullulanase-type a-amylase gene from Fig. 6. Rotational change of Trp356 and Tyr374 between unliganded TVA II and complex with 4 2 -P2. (A) Unliganded TVA II. (B) Structure of complex with 4 2 -P2. The 2F o –F c electron density maps contoured 1 r are shown for both models. Gln112 with the asterisk belongs to the MOL-2 molecule. Table 3. Kinetic parameters for starch and pullulan. k cat (s )1 ) K m (%) k cat /K m (s )1 Æ% )1 ) Starch Wild-type 120 ± 2.9 0.23 ± 0.010 520 ± 26 Y374A 9.4 ± 0.35 0.19 ± 0.020 40 ± 5.5 Pullulan Wild-type 140 ± 6.1 1.4 ± 0.12 100 ± 10 Y374A 4.8 ± 0.082 0.57 ± 0.084 8.4 ± 1.2 Ó FEBS 2004 Structure of T. vulgaris a-amylase II complex (Eur. J. Biochem. 271) 2537 Thermoactinomyces vulgaris R-47. Biosci. Biotechnol. Biochem. 57, 395–401. 5. Shimizu, M., Kanno, M., Tamura, M. & Suekane, M. (1978) Purification and some properties of a novel a-amylase produced byastrainofThermoactinomyces vulgaris. Agric. Biol. Chem. 42, 1681–1688. 6. Sakano, Y., Hiraiwa, S., Fukushima, J. & Kobayashi, T. (1982) Enzymatic properties and action patterns of Thermoactinomyces vulgaris a-amylase. Agric. Biol. Chem. 46, 1121–1129. 7. Kuriki, T., Okada, S. & Imanaka, T. (1988) New type of pull- ulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis. J. Bacteriol. 170, 1554–1559. 8. Tonozuka, T., Mogi, S., Shimura, Y., Ibuka, A., Sakai, H., Matsuzawa,H.,Sakano,Y.&Ohta,T.(1995)Comparisonof primary structures and substrate specificities of two pullulan- hydrolyzing a-amylases, TVA I and TVA II, from Thermoactino- myces vulgaris R-47. Biochim. Biophys. Acta 1252, 35–42. 9. Sakano, Y., Fukushima, J. & Kobayashi, T. (1983) Hydrolysis of a-1,4- and a-1,6-glucosidic linkages in trisaccharides by the Thermoactinomyces vulgaris a-amylases. Agric. Biol. Chem. 47, 2211–2216. 10. Ibuka, A., Tonozuka, T., Matsuzawa, H. & Sakai, H. (1998) Conversion of neopullulanase-a-amylase from Thermoactino- myces vulgaris R-47 into an amylopullulanase-type enzyme. J. Biochem. 123, 275–282. 11. Kamitori, S., Kondo, S., Okuyama, K., Yokota, T., Shimura, Y., Tonozuka, T. & Sakano, Y. (1999) Crystal structure of Thermo- actinomyces vulgaris R-47 a-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 A ˚ resolution. J. Mol. Biol. 287, 907–921. 12.Kamitori,S.,Abe,A.,Ohtaki,A.,Kaji,A.,Tonozuka,T.& Sakano, Y. (2002) Crystal structures and structural comparison of Thermoactinomyces vulgaris R-47 a-amylase1(TVAI)at1.6A ˚ resolution and a-amylase 2 (TVAII) at 2.3 A ˚ resolution. J. Mol. Biol. 318, 443–453. 13. Yokota, T., Tonozuka, T., Kamitori, S. & Sakano, Y. (2001) The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 a-amylase: effects of domain N on activity, specificity, sta- bility and dimerization. Biosci. Biotechnol. Biochem. 65, 401–408. 14. Yokota, T., Tonozuka, T., Shimura, Y., Ichikawa, K., Kamitori, S. & Sakano, Y. (2001) Structures of Thermo- actinomyces vulgaris R-47 a-amylase II complexed with substrate analogues. Biosci. Biotechnol. Biochem. 65, 619–626. 15. Kondo, S., Ohtaki, A., Tonozuka, T., Sakano, Y. & Kamitori, S. (2001) Studies on the hydrolyzing mechanism for cyclodextrins of Thermoactinomyces vulgaris R-47 a-amylase 2 (TVAII). X-ray structure of the mutant E354A complexed with b-cyclodextrin, and kinetic analysis on cyclodextrins. J. Biochem. 129, 423–428. 16. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 17. Kunkel, T.A., Roberts, J.D. & Zakour, R.A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382. 18. Ichikawa, K., Tonozuka, T., Yokota, T., Shimura, Y. & Sakano, Y. (2000) Analysis of catalytic residues of Thermoactinomyces vulgaris R-47 a-amylase II (TVA II) by site-directed mutagenesis. Biosci. Biotechnol. Biochem. 64, 2692–2695. 19. Kamitori, S., Satou, T., Tonozuka, T., Sakano, Y., Matsuzawa, H. & Okuyama, K. (1995) Crystallization and preliminary X-ray analysis of Thermoactinomyces vulgaris R-47 a-amylase II. J. Struct. Biol. 114, 229–231. 20. Tonozuka, T., Sakai, H., Ohta, T. & Sakano, Y. (1994) A con- venient enzymatic synthesis of 4 2 -a-isomaltosylisomaltose using Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II). Carbohydr. Res. 261, 157–162. 21. Rossman, M.G. & van Beek, C.G. (1999) Data processing. Acta Crystallog. Sect. D 55, 1631–1640. 22. Bru ¨ nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu,N.S.,Read,R.J.,Rice,L.M.,Simonson,T.&Warren, G.L. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. Sect. D 54, 905–921. 23. Bru ¨ nger, A.T. (1992) Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475. 24. Ramachandran, G.N. & Sasisekharan, V. (1968) Conformation of polypeptides and proteins. Adv. Protein Chem. 23, 283–437. 25. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1992) PROCHECK V.2: Programs to Check the Stereo- chemical Quality of Protein Structure. Oxford Molecular Ltd, Oxford, UK. 26. Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19–23. 27. Davies, G.J., Wilson, K.S. & Henrissat, B. (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem. J. 321, 557–559. 28. Matsuura, Y. (2002) A possible mechanism of catalysis involving three essential residues in the enzymes of a-amylase family. Biologia 57, 21–27. 29. Ohtaki, A., Kondo, S., Shimura, Y., Tonozuka, T., Sakano, Y. & Kamitori, S. (2001) Role of Phe286 in the recognition mechanism of cyclomaltooligosaccharides (cyclodextrins) by Thermo- actinomyces vulgaris R-47 a-amylase 2 (TVAII): X-ray structures of the mutant TVAIIs, F286A and F286Y, and kinetic analyses of the Phe286-replaced mutant TVAIIs. Carbohydrate Rese. 334, 309–313. 30. Hondoh, H., Kuriki, T. & Matsuura, Y. (2003) Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase. J. Mol. Biol. 326, 177–188. 31. Brzozowski, A.M. & Davies, G.J. (1997) Structure of the Asper- gillus oryzae a-amylase complexed with the inhibitor acarbose at 2.0 A ˚ resolution. Biochemistry 36, 10837–10845. 32. Lawson, C.L., van Montfort, R., Strokopytov, B., Rozeboom, H.J., Kalk, K.H., de Vries, G.E., Penninga, D., Dijkhuizen, L. & Dijkstra, B.W. (1994) Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J. Mol. Biol. 236, 590–600. 33. Kim, J.S., Cha, S.S., Kim, H.J., Kim, T.J., Ha, N.C., Oh, S.T., Cho,H.S.,Cho,M.J.,Kim,M.J.,Lee,H.S.,Kim,J.W.,Choi, K.Y., Park, K.H. & Oh, B.H. (1999) Crystal structure of a maltogenic amylase provides insights into a catalytic versatility. J. Biol. Chem. 274, 26279–26286. 34. Kuriki, T., Kaneko, H., Yanase, M., Takata, H., Shimada, J., Handa, S., Takada, T., Umeyama, H. & Okada, S. (1996) Con- trolling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydro- phobicity in active center. J. Biol. Chem. 271, 17321–17329. 35. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crys- tallog. 24, 946–950. 36. Merritt, E.A. & Murphy, M.E.P. (1994) Raster3d, A program for photorealistic molecular graphics. Version 2.0. Acta Crystallog. Sect. D, 50, 869–873. 2538 M. Mizuno et al. (Eur. J. Biochem. 271) Ó FEBS 2004 . The crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II (TVA II) complexed with transglycosylated product Masahiro Mizuno 1 ,. on the hydrolyzing mechanism for cyclodextrins of Thermoactinomyces vulgaris R-47 a-amylase 2 (TVAII). X-ray structure of the mutant E354A complexed with