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
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ABSTRACT |
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In order to study the interaction of soybean
-amylase with substrate, we solved the crystal structure of
-cyclodextrin-enzyme complex and compared it with that of
-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
-amylase and
formed the same crystals as the native enzyme. The crystal structure of
recombinant enzyme complexed with
-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-
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
-amylase. The conformation of
the bound
-cyclodextrin takes an ellipsoid shape in contrast to the
circular shape of the bound
-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
-amylase.
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INTRODUCTION |
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-Amylase (
-1,4-glucan maltohydrolase; EC 3.2.1.2) catalyzes
the removal of
-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
-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
-amylase) (10-12).
cDNAs of -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
-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
-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 -amylase complexed with
-cyclodextrin (
-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
-amylase at 2.3-Å resolution. The structural analysis of the soybean maltose-
-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
-CD·
-amylase and maltose·
-amylase complexes
revealed that a flexible loop plays a key role in the reaction.
CDs and maltose competitively inhibit the activity of -amylase by
binding to the active cleft (12, 20).
-CD binds to soybean
-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
-CD·
-amylase complex showed that only one glucose residue in
the
-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
-amylase on the
polymeric substrate (11, 21).
-CD is the cyclic oligosaccharide
consisting of seven glucoses, while
-CD has six glucoses in the
ring. The diameter of cavity in
-CD is about 1.2 times as long as
that in
-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
-CD (1-2 mM) is roughly
3 times that of
-CD (0.3-0.5 mM) (23-25), it should be
clarified whether the glucose residue involved in the binding of
-CD
still remains or whether the least favored interactions occur between
-CD and the enzyme.
In this study, the cDNA sequence of soybean -amylase was cloned
and expressed in E. coli. The crystal structure of the
recombinant
-amylase complexed with
-CD was analyzed at 2.07 Å to elucidate the flexibility of the substrate binding site.
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EXPERIMENTAL PROCEDURES |
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Cloning and Sequencing of Soybean -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
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
-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.
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 -Amylase--
DNA
sequences coding a mature
-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 -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-
-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--
-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 -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-
-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
-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 -Amylase--
The purified
recombinant enzyme was subjected to crystallization under similar
conditions as for native soybean
-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 -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 2
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
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 -amylase
complexed with
-CD (12). After rigid body refinement, three amino
acids were substituted (as shown in Fig. 1), and atoms in glucose
residues 3-5 of
-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
-CD
atoms. The model was fitted as judged by inspection of electron density maps calculated with both 2Fo
Fc and Fo
Fc coefficients.
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RESULTS |
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Cloning of Soybean -Amylase cDNA--
We screened cDNAs
encoding
-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
-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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Expression of Soybean -Amylase cDNA in E. coli--
When
-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-
-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
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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Purification and Characterization of Recombinant
-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
-amylase deduced from the
nucleotide sequence of the cDNA and indicated that the
-amino
group of the N-terminal alanine residue of the recombinant enzyme is
not acetylated, although the native enzyme is acetylated (36).
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Quality of Refined Structure--
The refined structure of
recombinant -amylase complexed with
-CD had an
R-factor of 0.158 and a free R-factor of 0.211, when all observed data upon 2
(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
-CD molecule. The position of
-CD in active cleft is similar to
that of
-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
-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
-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 -CD and the Native Enzyme Complexed with
-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
-amylase (12). The rigid body fitting indicated an
r.m.s. distance of 0.22 Å between pairs equivalent C-
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-
-C-
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
-amylase complexed with
-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
-amylase is reduced, while
that in the native enzyme complexed with
-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
-amylase by means
of x-ray crystallography and protein engineering.
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The Structure of Bound -CD--
At the beginning of refinement,
the electron density for sugar units of
-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
- and
-CDs in the active cleft of the enzyme. While
-CD bound
to
-amylase took a flat circular form (12), the conformation of the
bound
-CD in the
-amylase complex was found to be a distorted ellipse. The distorted shape of the bound
-CD was dissimilar to the
shape observed in the complex of the maltose-binding protein (39) and
to that observed in crystalline
-CD hydrate (40). This suggests that
the conformation of the bound
-CD changed to fit the enzyme rather
than vice versa.
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Interactions of -CD with Enzyme--
Interactions between the
bound
-CD and the recombinant
-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
-CD and protein. In these interactions, water-mediated hydrogen bonds with H2O662 were
found only in the
-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
-amylase with
-CD. Interactions with Van der Waals contact were
similar to those of the
-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
-CD·
-amylase complex (12).
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DISCUSSION |
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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
-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
-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
-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
-amylases sequenced so far (12). The latter residue was found
to be arginine in the protein sequence of soybean
-amylase (43). The
three-dimensional structure of
-amylase complexed with
-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-
1 and N-
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-
atoms form water-mediated hydrogen bonds with O-
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 -CD·
-amylase complex and
compared it with that of
-CD complex (12) in order to investigate interactions between
-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
-CD has one more
glucose residue than
-CD affects the increase of the
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
- and
-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, -amylase and
animal
-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
-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
-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
-1,4-glycosidic linkage (step 1). The
bound substrate is hydrolyzed by the two catalytic residues of
Glu186 and Glu380 in
-amylase (16). The
enzymatic reaction produces
-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
-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
-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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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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* 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.
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.
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