Crystal Structure of Recombinant Soybean beta -Amylase Complexed with beta -Cyclodextrin*

Motoyasu Adachi, Bunzo MikamiDagger , Tomoyuki Katsube§, and Shigeru Utsumi

From the Research Institute for Food Science, Kyoto University, Uji Kyoto 611-0011, Japan and the § Department of Domestic Science, Shimane Prefectural Shimane Women's College, Shimane 690-0044, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In order to study the interaction of soybean beta -amylase with substrate, we solved the crystal structure of beta -cyclodextrin-enzyme complex and compared it with that of alpha -cyclodextrin-enzyme complex. The enzyme was expressed in Escherichia coli at a high level as a soluble and catalytically active protein. The purified recombinant enzyme had properties nearly identical to those of native soybean beta -amylase and formed the same crystals as the native enzyme. The crystal structure of recombinant enzyme complexed with beta -cyclodextrin was refined at 2.07-Å resolution with a final crystallographic R value of 15.8% (Rfree = 21.1%). The root mean square deviation in the position of C-alpha atoms between this recombinant enzyme and the native enzyme was 0.22 Å. These results indicate that the expression system established here is suitable for studying structure-function relationships of beta -amylase. The conformation of the bound beta -cyclodextrin takes an ellipsoid shape in contrast to the circular shape of the bound alpha -cyclodextrin. The cyclodextrins shared mainly two glucose binding sites, 3 and 4. The glucose residue 4 was slightly shifted from the maltose binding site. This suggests that the binding site of the cyclodextrins is important for its holding of a cleaved substrate, which enables the multiple attack mechanism of beta -amylase.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

beta -Amylase (alpha -1,4-glucan maltohydrolase; EC 3.2.1.2) catalyzes the removal of beta -anomeric maltose from the nonreducing ends of starch and glycogen. This enzyme is distributed in higher plants and in some microorganisms. The cDNAs from five kinds of plants (soybean (1), barley (2), rye (3), Arabidopsis thaliana (4), and sweet potato (5)) and those of three kinds of bacterium (Bacillus polymixa (6, 7), Bacillus circulans (8), and Clostridium thermosulfurogenes (9)) have been cloned and sequenced. Plant beta -amylases are similar to each other in terms of their physicochemical properties, i.e. molecular mass (50-60 kDa), optimum pH, amino acid sequence, and their subunit structure (with the exception of the homotetramer sweet potato beta -amylase) (10-12).

cDNAs of beta -amylase from barley, sweet potato, and soybean have been expressed in E. coli (1, 5, 13). Yoshigi et al. (14) tried to produce the thermostable barley beta -amylase by random and site-directed mutagenesis using the E. coli expression system. The produced 7-fold mutant was more stable than the wild-type recombinant enzyme by 11.6 °C (14). The soybean beta -amylase was expressed using pKK233-2 expression vector (1). The catalytic efficiency of the recombinant enzyme, however, was lower than that of the native enzyme.

The crystal structure of native soybean beta -amylase complexed with alpha -cyclodextrin (alpha -CD)1 was solved at 2.0 Å (12) by the isomolphous replacement method. Cheong et al. (15) reported the crystal structure of tetrameric sweet potato beta -amylase at 2.3-Å resolution. The structural analysis of the soybean maltose-beta -amylase complex indicated that Glu186 and Glu380 play important roles in the enzymatic reaction as general acid and base catalysts, respectively (16). This finding is supported by the results of site-directed mutagenesis (17, 18) and affinity labeling (19). In addition, the structures of alpha -CD·beta -amylase and maltose·beta -amylase complexes revealed that a flexible loop plays a key role in the reaction.

CDs and maltose competitively inhibit the activity of beta -amylase by binding to the active cleft (12, 20). alpha -CD binds to soybean beta -amylase of an open loop form (12), whereas two maltose molecules tandem bind within the active cleft of the enzyme in a closed loop form (16). These maltose binding sites are located on both sides of the catalytic residues and are postulated to be substrate binding sites, subsites 1 and 2 and subsites 3 and 4 (16). The structure of the alpha -CD·beta -amylase complex showed that only one glucose residue in the alpha -CD binds near subsite 4, where the binding force essentially involves hydrophobic interactions (16). The exact position of this glucose residue is shifted about one-half residue to the side of the reducing end, suggesting the flexibility of subsite 4 against altered positions of glucose residue (16). This flexibility of subsite 4 may elucidate the mechanism of single chain attack of beta -amylase on the polymeric substrate (11, 21). beta -CD is the cyclic oligosaccharide consisting of seven glucoses, while alpha -CD has six glucoses in the ring. The diameter of cavity in beta -CD is about 1.2 times as long as that in alpha -CD, and the bond angle and two torsion angles in the glycosidic links differ slightly between the two CDs (22). Since the Ki value of beta -CD (1-2 mM) is roughly 3 times that of alpha -CD (0.3-0.5 mM) (23-25), it should be clarified whether the glucose residue involved in the binding of alpha -CD still remains or whether the least favored interactions occur between beta -CD and the enzyme.

In this study, the cDNA sequence of soybean beta -amylase was cloned and expressed in E. coli. The crystal structure of the recombinant beta -amylase complexed with beta -CD was analyzed at 2.07 Å to elucidate the flexibility of the substrate binding site.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Sequencing of Soybean beta -Amylase cDNA-- Poly(A) RNA was prepared from the developing cotyledons of soybean (Glycine max L. cv. Wasesuzunari) using an EXTRACT-A-PLANT RNA isolation kit (CLONTECH) and mRNA purification kit (Amersham Pharmacia Biotech). A cDNA library was constructed in lambda  ZAPII phage using a ZAP-cDNA synthesis kit (Stratagene) (26). The cDNA library was screened with the synthesized oligonucleotide Primer N (5'-GCCACTTCCGACAGTAACATGC), referring to the nucleotide sequence of beta -amylase reported for soybean cv. Bonminori (1). Plaque hybridization was performed with 5'-end-labeled primer N in 6 × SSC at 50 °C for 12 h. Nylon membranes (HybondN, Amersham) were washed with 6 × SSC at 65 °C. lambda  ZAPII phages containing full-length cDNA were screened by restriction enzyme (EcoRI and XhoI) analysis, and six clones were selected. Six plasmids containing full-length cDNA were excised with the R408 helper phage, and one (pBSB7) of the resultant plasmids containing the longest cDNA was sequenced by Taq dideoxy cycle sequencing using Applied Biosystems sequencer model 373A.

Construction of an Expression Plasmid for beta -Amylase-- DNA sequences coding a mature beta -amylase were amplified by polymerase chain reaction using primers N and C (5'-CGCGGATCCAAGCTTGGGAAATCAACCATCAACTTTC) containing a BamHI site. Polymerase chain reaction was conducted on pBSB7 with reagents supplied in kit form (Takara Shuzo Ltd., Kyoto, Japan) in a DNA thermal cycler model 480 (Perkin-Elmer). A polymerase chain reaction cycle consisted of denaturation at 95 °C for 30 s, annealing at 50 °C for 1 min, and extension at 72 °C for 3 min. After 30 cycles, the products were separated by electrophoresis on 1.2% (w/v) agarose gel and purified by using glass powder (Takara). The resultant 1.5-kilobase pair fragment was blunted by a blunting kit (Takara), cut with BamHI at the 3'-part, and inserted between the filled in NcoI and BamHI sites of an expression vector pET21d (Novagen, Madison, WI) to generate pESBA. The blunted 1.5-kilobase pair fragment was inserted into the filled in NcoI site of an expression vector pKK233-2 (Amersham Pharmacia Biotech) to generate pKSBA. The cDNA sequence in the pKSBA was resequenced.

Expression and Detection of beta -Amylase from E. coli-- The expression plasmid pESBA was transformed into E. coli strain BL21(DE3), BL21(DE3)pLysS, HMS174(DE3), and HMS174(DE3)pLysS, and pKSBA into JM105, JM107, JM109, MV1184, SOLR, and XLI-Blue. Each E. coli strain harboring individual expression plasmid was cultured in LB, TB, 2× YT, NZY, and M9 minimal medium (M/C), each supplemented with ampicillin (50 µg/ml) at 20, 25, 30, and 37 °C for 3, 24, and 40 h. At A600 = 0.3, 0.8, or 1.5, isopropyl-beta -D-thiogalactopyranoside was added to a final concentration of 1 mM. The cells were harvested by centrifugation and disrupted by sonication in 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The cell debris and the supernatant were fractionated by centrifugation. The SDS-polyacrylamide gel electrophoresis analysis of the total cells, the cell debris, and the supernatant was performed according to the method of Laemmli (27) as described previously (28). The proteins separated in SDS gels were stained by Coomassie Brilliant Blue R-250.

Assay Methods-- beta -Amylase activity was measured in 0.1 M acetate buffer, pH 5.4, according to the Bernfeld method (29) as described by Morita et al. (30). One unit of activity is defined as the amount of enzyme to produce 1 µmol of maltose/min at 37 °C. Potato amylopectin was used as a substrate. Protein amounts of the purified and crude enzymes were determined by an extinction coefficient of 97 mM-1·cm-1 at 280 nm (31) and by the method of Bradford (32) with bovine serum albumin as a standard, respectively. The dependence of enzyme activity on pH was evaluated with 0.05 M buffers containing Tris, citrate, phosphate, and borate. The pH and ionic strength were adjusted with NaOH and NaCl, respectively. Thermal stability was determined by the remaining activity after incubation at temperatures from 30 to 80 °C for 30 min. The value of T50 was defined as the temperature indicating 50% residual activity. Isozyme II from soybean (native enzyme) was isolated and purified as described by Mikami et al. (33).

Bench Scale Expression and Purification of beta -Amylase-- Four hundred ml of LB medium in a 2-liter flask (total of 12 liters) was inoculated with 4 ml (× 30) of an overnight grown culture of JM105 harboring pKSBA and cultured at 37 °C. At A600 = 0.8, the medium was cooled down to 20 °C, and isopropyl-beta -D-thiogalactopyranoside was added to a final concentration of 1 mM. After cultivation for 40 h at 20 °C, the induced cells were harvested by centrifugation. The cells were suspended in the extraction buffer and disrupted by sonication at 4 °C. After the addition of ME to a final concentration of 50 mM and centrifugation at 10,000 × g for 20 min, the resultant crude extract was fractionated with a 35% saturation of ammonium sulfate. The supernatant collected by centrifugation was applied to a butyl-Toyopearl column (4 × 25 cm) previously equilibrated with 50 mM sodium phosphate buffer (pH 6.5) containing 5 mM EDTA, 18 mM ME, and 30% saturated ammonium sulfate. Proteins were eluted with a descending gradient of ammonium sulfate ranging from 30 to 0%. The fractions containing beta -amylase activity were pooled and dialyzed against 0.05 M sodium acetate buffer (pH 4.8) containing 1 mM EDTA and 18 mM ME. After dialysis, the protein solution was applied to a CM-Sephadex C-50 column (2 × 25 cm) (33). Proteins were eluted with a pH gradient of 50 mM sodium acetate buffer from 4.8 to 6.0. To investigate the homogeneity and pI of the purified recombinant enzyme, polyacrylamide gel isoelectric focusing was performed using Ampholine pH3-10 (Amersham Pharmacia Biotech).

N-terminal Amino Acid Sequence Analysis-- The N-terminal amino acid sequence analysis was performed by automatic Edman degradation on an Applied Biosystems model 477A pulse-liquid sequencer system. The purified recombinant protein in buffer solution was dialyzed against distilled water and applied to a polybrene-treated glass membrane.

Crystallization of Recombinant beta -Amylase-- The purified recombinant enzyme was subjected to crystallization under similar conditions as for native soybean beta -amylase (12) by the vapor diffusion method using Linbro multiwell tissue culture plates (34). The crystals were grown at a constant temperature of 4 °C from an initial protein concentration of 10 mg/ml in a drop composed of an equal volume (5 µl each) of a protein solution and a well solution (1 ml) containing 49% ammonium sulfate, 0.1 M sodium acetate buffer, 1 mM EDTA, and 18 mM ME, pH 5.4.

Data Collection-- Crystals formed by the hanging drop method were transferred from the drop to 0.1 M pH 5.4 sodium acetate buffer containing 1 mM EDTA and 50% saturated ammonium sulfate for storage. The crystals belong to space group P3121 with unit cell dimensions a = b = 86.03 and c = 144.80 (Z = 6) and grow to a size of about 0.4 × 0.5 × 0.4 mm. The crystals were soaked in the same buffer containing 5 mM beta -CD for 1 h at room temperature before data collection. Data were collected on a RIGAKU R-AXISIIc imaging plate area detector at the Institute for Chemical Research, Kyoto University. The detector was positioned at a distance of 110 mm from the crystal at a 2theta angle of 7°. The crystal was exposed to x-rays for 15 min in oscillation frame of 1.5°. The crystal diffracted the x-rays to 2.07 Å and was stable during 12 h for 40 oscillation frames. The cell dimensions were refined by the least square method. Data from different frames were integrated separately and then merged together (Rmerge = 6.0% for 152,726 measurements). In the resolution range of 10-2.07 Å, 36,696 of the 39,214 theoretically possible reflections having intensities of more than 2 sigma  were used for the refinement.

Model Building and Refinement-- Structure was refined using the program X-PLOR version 3.1 (35). The initial phases were calculated using the coordinates from the protein structure of native beta -amylase complexed with alpha -CD (12). After rigid body refinement, three amino acids were substituted (as shown in Fig. 1), and atoms in glucose residues 3-5 of beta -CD were added to the model. The model was systematically improved throughout iterative cycles containing positional and B-factor refinements. The model was rebuilt using the Turbo-Frodo program (BioGraph) and improved using a stepwise increased resolution of data and the addition of solvent and remaining beta -CD atoms. The model was fitted as judged by inspection of electron density maps calculated with both 2Fo - Fc and Fo - Fc coefficients.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of Soybean beta -Amylase cDNA-- We screened cDNAs encoding beta -amylase from a soybean cDNA library. The first screening gave 200 positive clones from 80,000 plaques. Six phages containing a full-length cDNA were selected from 20 positive clones by restriction enzyme analysis after a second successive screening. The inserts from six positive clones were in the range of 1.6-1.7 kilobase pairs. The longest cDNA was sequenced. The cDNA had an open reading frame with 1488 bp coding a polypeptide composed of 496 amino acids with a calculated molecular mass of 56,069 Da. Comparison of the nucleotide sequence of beta -amylase cDNA with that from cv. Bonminori (1) shows that two nucleotide substitutions occur in the coding region, resulting in two replacements of amino acid residues. The 202nd arginine and 399th lysine were replaced by glycine and arginine, respectively, in Wasesuzunari (Fig. 1).


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Fig. 1.   Differences among the amino acid sequences of soybean beta -amylase. The terminator is indicated by three asterisks. The differences in sequence are indicated by underlining. The cDNA sequence from cv. Bonminori is reported by Totsuka et al. (1).

Expression of Soybean beta -Amylase cDNA in E. coli-- When beta -amylase cDNA was expressed in E. coli at 37 °C, the expression level of active enzyme was only 0.5 and 1% of total E. coli proteins in the cases of pKSBA in JM105 and pESBA in BL21(DE3), respectively. Thus, we tried to establish conditions giving a high level and stable expression by changing E. coli strain, culture temperature, and induction timing and period, as described under "Experimental Procedures." Although we were unable to induce a high level and stable expression under any conditions in the case of pESBA, we succeeded at doing so in the case of pKSBA under the following conditions: strain JM105 cells harboring pKSBA grown at 37 °C in LB and expression induced at A600 = 0.8 by isopropyl-beta -D-thiogalactopyranoside at 20 °C for 40 h. SDS-polyacrylamide gel electrophoresis analysis and specific activity of the extract showed that the expression level was approximately 10% of cellular protein (Fig. 2A, lane 1). Degradation of the expressed protein decreased dramatically compared with that in the case of induction at 37 °C (data not shown), and more than 90% of this protein was recovered in soluble fraction after sonication. The activity of the recombinant enzyme extracted from 1 ml of growth medium was 34.7 units, which was 37.3 times that obtained by Totsuka et al. despite the similar expression conditions (1). In addition, the activity of the purified enzyme was about 3 times as high as that they reported. The cDNA sequence in the pKSBA differed from the cloned cDNA at two positions as a result of the polymerase chain reaction (Fig. 1). One replacement resulted in an amino acid substitution at position 76 (Phe right-arrow Leu), as shown in Fig. 1. Since the substitution was regarded as causing no effective change in enzyme properties based on a reasonable specific activity of crude extract, we used pKSBA for further analysis.


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Fig. 2.   SDS-polyacrylamide gel electrophoresis (A) of fraction from E. coli JM105 and gel isoelectric focusing (B) of beta -amylase purified form E. coli. A, lane 1, homogenate (5 µg); lane 2, crude extract (5 µg); lane 3, fraction eluted from the butyl-Toyopearl column (2 µg); lane 4, fraction eluted from the CM-Sephadex column (1.5 µg). B, lane 1, native soybean beta -amylase; lane 2, purified recombinant beta -amylase.

Purification and Characterization of Recombinant beta -Amylase-- E. coli strain JM105 cells harboring expression plasmid pKSBA were used for bench scale preparation of the recombinant enzyme. Seventy g of the wet bacteria were recovered from 12 liters of culture medium. The recombinant enzyme was purified by hydrophobic and ion exchange chromatography. This procedure resulted in 13.5-fold purification and 31% recovery and provided 150 mg of purified enzyme. The enzyme was found to be homogeneous on both SDS-polyacrylamide gel electrophoresis (Fig. 2A, lane 4) and gel isoelectric focusing (Fig. 2B, lane 2), and had a molecular mass of 56 kDa and a pI value of 5.4, which is higher than that (5.25) of the native enzyme (isozyme 2) (33). The N-terminal amino acid sequence was determined to be Ala-Thr-Ser-Asp-Ser-Asn-Met-Leu-Leu-Asn-Tyr-Val. This was consistent with the N-terminal sequence of the mature beta -amylase deduced from the nucleotide sequence of the cDNA and indicated that the alpha -amino group of the N-terminal alanine residue of the recombinant enzyme is not acetylated, although the native enzyme is acetylated (36).

The catalytic characteristics of the recombinant beta -amylase were compared with those of the native soybean enzyme (Table I). The values of Km, Vmax, optimum pH, the inhibition constant (Ki) for alpha -and beta -CDs, and half inactivation temperature (T50) for recombinant enzyme were nearly identical to those for the native enzyme.

                              
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Table I
Comparison of enzymatic properties for the native and recombinant enzyme

Quality of Refined Structure-- The refined structure of recombinant beta -amylase complexed with beta -CD had an R-factor of 0.158 and a free R-factor of 0.211, when all observed data upon 2 sigma  (34,127 reflections) between 10.0 and 2.07 Å were included. The r.m.s. deviations of bond lengths, bond angles, dihedral angles, and improper angles were 0.013 Å, 2.89°, 24.3°, and 1.11°, respectively. The final model contains 490 amino acids (residues 6-495), 318 water molecules, a sulfate ion, and a beta -CD molecule. The position of beta -CD in active cleft is similar to that of alpha -CD (12). The five amino acids of the N terminus were missing from the model because of their disorder. The ramachandran plot of the refined recombinant beta -amylase generated by the program PROCHECK (37) showed that 89.3% of the main-chain dihedral angles fell within the most favored regions. Only one residue of Arg420 was found in a disallowed region as reported in alpha -CD complex (12). The mean positional errors were estimated to be about 0.18 Å from Luzzati plots (38). The average temperature factors for protein, solvent, and all atoms were 20.6, 36.6, and 22.3 Å2, respectively.

Comparison of Structures between the Recombinant Enzyme Complexed with beta -CD and the Native Enzyme Complexed with alpha -CD-- To confirm the fidelity of the expression system constructed here, we compared the protein structure of the recombinant enzyme with that of native soybean beta -amylase (12). The rigid body fitting indicated an r.m.s. distance of 0.22 Å between pairs equivalent C-alpha atoms. This indicates that the native and recombinant enzyme have the same overall structure. Fig. 3 provides a more detailed comparison. From the plot, three peaks were formed at around Ile102, Gly202, and Thr342. The C-alpha -C-alpha distances at Ile102, Gly202, and Thr342 were 1.06, 0.74, and 1.83 Å, respectively. The first region around Ile102 is included in the flexible loop having a high B-factor value. The average B value of atoms in residues 96-103 was 40.4Å2. The second is the region around the revised Gly202, which was modeled as arginine in the structure of beta -amylase complexed with alpha -CD (12). The displacement resolved the discrepancy observed between the model and electron density during refinement for the crystal structure of the native enzyme. The third region includes Cys343. The SH group of the residue in the recombinant beta -amylase is reduced, while that in the native enzyme complexed with alpha -CD is modified by ME to form a mixed disulfide (12). Therefore, these three differences can be discounted, and the structure and enzymatic characteristics described above indicate that the expression system established here is suitable for studies of structure-function relationships of beta -amylase by means of x-ray crystallography and protein engineering.


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Fig. 3.   Plot of C-alpha distances between recombinant beta -amylase·beta -CD and native beta -amylase·alpha -CD complexes versus residue number. A least-square rigid body fitting was performed by the program of Turbo-Frodo.

The Structure of Bound beta -CD-- At the beginning of refinement, the electron density for sugar units of beta -CD was clear and unambiguous in only three glucose residues, 2, 3, and 4, but all seven glucose residues were visible after refinement. Fig. 4 shows an omit map and the structures of beta - and alpha -CDs in the active cleft of the enzyme. While alpha -CD bound to beta -amylase took a flat circular form (12), the conformation of the bound beta -CD in the beta -amylase complex was found to be a distorted ellipse. The distorted shape of the bound beta -CD was dissimilar to the shape observed in the complex of the maltose-binding protein (39) and to that observed in crystalline beta -CD hydrate (40). This suggests that the conformation of the bound beta -CD changed to fit the enzyme rather than vice versa.


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Fig. 4.   Stereo views of the CD binding sites of soybean beta -amylase. A, Fo - Fc omit map contoured at 3 sigma  for beta -CD molecule bound in the active site cleft. B, hydrogen bond interactions of beta -amylase with beta -CD. The protein residues of each complex were superimposed using the Rigid Program of Turbo-Frodo. Thick lines represent the beta -CD structure in the beta -CD complex. Thin lines represent the alpha -CD molecule in the alpha -CD complex. Residue types and sequence numbers are labeled. The water molecules in the complex with beta -CD are indicated by closed circles. Dotted lines represent potential hydrogen bond interactions within 3.2 Å. Protein residues having apparent Van der Waals contact with the beta -CD are labeled.

The following parameters (average and S.E.) were found for the seven D-glucose residues in the bound beta -CD: glycosidic bridge atom angle, 118.9 ± 0.8°; phi  torsion angle (O-4 ... C-1-O-4'-C-4'), +166.4 ± 4.5°; psi  torsion angle (C-1-O-4'-C-4' ... O-4'), -170.2 ± 4.3°. The S.E. for the phi  torsion angle was 1.8 times higher than that of alpha -CD (14) in the alpha -CD·beta -amylase complex, although that for the psi  angle was almost the same. Moreover, we compared phi  and psi  angles in each glycosidic linkage of beta -CD with those of alpha -CD by plotting (Fig. 5). The profiles of the phi  and psi  angles were similar to each other except for the phi  angle between glucose residues 4 and 5. The difference of this phi  torsion angle was 30°, and for both residues the angle still corresponded to a low energy conformation (41).


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Fig. 5.   Dihedral angles of phi  and psi  in glycosidic linkage between glucose residues in bound beta -CD. Closed and open symbols represent the angles in beta -CD and alpha -CD, respectively. Square and circular symbols represent phi  and psi  angles, respectively. The residue number is indicated in Fig. 4.

Interactions of beta -CD with Enzyme-- Interactions between the bound beta -CD and the recombinant beta -amylase are summarized in Table II. There were four direct hydrogen bonds and five water-mediated hydrogen bonds by three water molecules between hydroxyl groups of beta -CD and protein. In these interactions, water-mediated hydrogen bonds with H2O662 were found only in the beta -CD complex, although direct hydrogen bonds with O atom of Ala382 and N atom of His300 and water-mediated hydrogen bonds with H2O655 and H2O755 were also found in the complex of beta -amylase with alpha -CD. Interactions with Van der Waals contact were similar to those of the alpha -CD complex (12), and there were the greatest number of contacts between glucose residue 4 and the protein. Leu383 formed an inclusion complex such as that in the alpha -CD·beta -amylase complex (12).

                              
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Table II
Van der Waals contacts and hydrogen bonds (<= 3.3 Å) between the beta -amylase and beta -CD

For comparison of the location of glucose residues, we calculated five r.m.s. distances in a residue between both CDs after C-alpha rigid body fitting for the protein. These r.m.s. values were 1.35, 0.84, 0.43, 0.33, and 2.4 Å for residues 1-5, respectively. Residue 4 allowed the best superimposition, and was located one-half residue from subsite 4 in the direction of the reducing end. The r.m.s. values of residues 4-1 gradually increased relative to that from 4 to 5. These results are consistent with the dramatic change of dihedral angle phi  between residues 4 and 5 (Fig. 5). This is probably because beta -CD forms water-mediated hydrogen bonds with H2O662, unlike the alpha -CD complex.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In general, the expression level of a foreign protein in E. coli depends on the kind of promoter, the stabilities of mRNA and the protein product, the culture conditions, and other such factors (42). To obtain a high level expression of recombinant soybean beta -amylase, various experimental conditions were studied. The results indicated that the induction temperature and period were the most important factors for high level expression of soybean beta -amylase. The system established here provided a recombinant enzyme having properties nearly identical to those of the native enzyme. Although our expression system was similar to that reported by Totsuka et al. (1), both the purified recombinant enzyme in this study and the native enzyme exhibited 3 times higher specific activity than that of the recombinant enzyme reported by these previous authors. As for the reasons why their recombinant enzyme had a much lower specific activity than the native enzyme, they proposed the following three factors: (i) the requirement of a molecular chaperon for correct folding; (ii) the requirement of N-terminal acetylation for activity; and (iii) the microheterogeneity of the native enzyme from soybean seed (1). Comparing our recombinant enzyme with the native enzyme, we found no significant difference in catalytic characteristics or protein structures, indicating that soybean beta -amylase does not require N-terminal acetylation and molecular chaperons other than those of E. coli for its activity and folding. The cDNA used in this study differs in two positions from that reported by Totsuka et al. (1). Gly202 and Arg399 in the cDNA from cv. Wasesuzunari were substituted with arginine and lysine, respectively, in the cDNA from cv. Bonminori. Gly202 and Arg399 are conserved in all other plant beta -amylases sequenced so far (12). The latter residue was found to be arginine in the protein sequence of soybean beta -amylase (43). The three-dimensional structure of beta -amylase complexed with beta -CD shows that Gly202 is about 10 Å from the catalytic residues near the molecular surface, and the substitution of Gly202 for arginine should cause steric hindrance with Tyr238 and Asn239. On the other hand, the atoms of N-eta 1 and N-eta 2 in Arg399 (about 20 Å from the active center) form direct hydrogen bonds with O atom in Asn10 (2.9 Å), O atom in Met441 (2.9 Å), and O atom in Lys44 (2.8 Å), and N-epsilon atoms form water-mediated hydrogen bonds with O-delta 1 in Asp490. The substitution of Arg399 for lysine may distort the protein structure due to a breakdown of the hydrogen network. The two substitutions mentioned above were probably the reason for the higher expression level and specific activity of the recombinant enzyme in the system constructed here relative to those reported by Totsuka et al. (1). The substitution at position 202 may have been particularly critical in this regard.

We have determined the structure of beta -CD·beta -amylase complex and compared it with that of alpha -CD complex (12) in order to investigate interactions between beta -amylase and its substrates. Our results indicated no significant differences between the two protein structures. Two CD molecules bind to the enzyme with very similar interactions mainly at glucose residues 3 and 4, and the residue 4 was the most readily superimposed. The fact that beta -CD has one more glucose residue than alpha -CD affects the increase of the phi  angle between glucose residues 4 and 5. We suggest that the interactions at the glucose residues 3 and 4 are not so much rigid but rather work to hold a substrate in the successive reaction because glucose residue 4 in the alpha - and beta -CDs is positioned about one-half residue from subsite 4 in the direction of the reducing end.

Generally, amylases cleave substrates by a pathway in which they randomly encounter a substrate chain (12). However, beta -amylase and animal alpha -amylase degrade polymeric substrates to products sequentially after complexing with the substrates in addition to degrading them randomly. This is commonly called a multiple attack (11, 44, 45) and allows beta -amylase to react efficiently. Here, we suggest that the binding site of CDs plays an important role in the action of the enzyme to retain the cleaved polymeric substrate (Fig. 6). This would explain the mechanism of this intriguing reaction of beta -amylase as follows; after a polymeric substrate, such as amylose, binds to each subsite of the enzyme from the nonreducing end, the flexible loop immediately closes. In this binding, the terminal maltose unit of the substrate (glucose residues 1 and 2) reverses by torsion in alpha -1,4-glycosidic linkage (step 1). The bound substrate is hydrolyzed by the two catalytic residues of Glu186 and Glu380 in beta -amylase (16). The enzymatic reaction produces beta -anomeric maltose and a new substrate that is destined to be cleaved in the next step. The flexible loop then opens, resulting in the exposure of the active site to solvent and the departure of the produced beta -maltose (step 2). For the next step, shift of the substrate occupying subsites 3 and 4 is required because the terminal maltose unit of the substrate has to reverse before binding to the subsites 1 and 2. The cleaved substrate is released from the subsites and held on the surface of the enzyme for the successive reaction. Furthermore, occupation of the CD-binding site by cleaved substrate fixes the flexible loop in an open conformation (12), which would make it easy to take the substrate in subsites 1 and 2. After all, the holding of the cleaved substrate at the CD binding site leads to a single chain attack (the slipping mechanism) (12) (step 3), whereas the release of the cleaved substrate into solvent leads to a multichain attack. The flexible binding of glucose around subsite 4 by hydrophobic interactions enables the slipping mechanism (16). Leu383, which forms an inclusion complex, is one of the residues that contribute to the slipping mechanism (12). Totsuka et al. (18) suggested by site-directed mutagenesis that Leu383 may work to bind polymeric substrate as a winder (18). Probably, Leu383 plays an important role in the single chain attack mechanism as well as in the binding of polymeric substrate, because the residue seems to block the cleaved substrate from release into the solvent (see Fig. 6). In addition, the side chain of His300 interacts with the glucose residue on subsite 4 or the residue 4 in CDs via hydrogen bonds. Since the extent of multiple attack can be varied by changing the pH (21, 46, 47), the residue may also play a key role in the multiple attack. Attempts to reveal the detailed mechanism of beta -amylase using x-ray structure analysis and enzymatic kinetics with the mutant enzymes obtained from the present system are currently in progress.


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Fig. 6.   Stereo view of a hypothetical single chain attack mechanism of beta -amylase. Thick lines indicate the substrate and produced maltose. The structures in steps 1 and 2 and in step 3 are drawn on the basis of the structure of maltose-beta -amylase complex (16) and beta -CD·beta -amylase complex, respectively. Numerals 1-4 and italicized numerals 3-6 show glucose residues on the four subsites (16) and those in beta -CD complexed with enzyme, respectively. Broken and thin lines indicate the flexible loop composed of protein residues from Gly96 to Ile103 and other protein residues around the active site, respectively.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Hiroaki Kato and Yasuo Hata of the Institute for Chemical Research (Kyoto University) for technical advice. Computation time was provided by the Super-Computer Laboratory (Institute for Chemical Research, Kyoto University).

    FOOTNOTES

* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D50866.

The atomic coordinates and structure factors(1bfn) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger To whom correspondence should be addressed. Tel.: 81-774-38-3763; Fax: 81-774-38-3764; E-mail: mikami{at}soya.food.kyoto-u.ac.jp.

1 The abbreviations used are: CD, cyclodextrin; r.m.s., root mean square; ME, 2-mercaptoethanol.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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