Purification, characterization, immunolocalization and structural
analysis of the abundant cytoplasmic b-amylase from
Calystegia
sepium
(hedge bindweed) rhizomes
Els J. M. Van Damme
1
, Jialiang Hu
1
, Annick Barre
2
, Bettina Hause
3
, Geert Baggerman
4
, Pierre Rouge
´
2
and
Willy J. Peumans
1
1
Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Leuven, Belgium;
2
Institut de Pharmacologie et
Biologie Structurale, Unite
´
Mixte de Recherche Centre National de la Recherche Scientifique 5089, Toulouse, France;
3
Institute of Plant
Biochemistry, Halle, Germany;

mays ) also contain, besides the classical abundant and
highly active endosperm b-amylases, low levels of another
so-called ‘tissue-ubiquitous’ form in leaves and roots [1].
b-Amylases have also been identified in roots of alfalfa
(Medicago sativa ) and several other forage legumes
including sweetclover (Melilotus officinalis ), red clover
(Trifolium pratense ), birdsfoot trefoil (Lotus corniculatus )
[4], and in pea (Pisum sativum ) epicotyls [5]. In addition,
b-amylases have been identified in species of the families
Solanaceae (potato, Solanum tuberosum ) [6] and Brassica-
ceae (Arabidopsis thaliana and Streptanthus tortuosus )
[7,8].
Extensive enzymatic studies of several b-amylases
unambiguously demonstrated that these enzymes exclu-
sively catalyze the release of b-maltose from the
nonreducing ends of a-1,4-linked oligo- and polyglucans.
Accordingly, b-amylases are believed to be involved in the
degradation of starch in the plant and/or a-1,4-linked
oligoglucans. Though this presumed role might hold true for
some b-amylases, it certainly cannot be extrapolated to all
plant b-amylases because (a) some b-amylases occur in
tissues that are devoid of starch, (b) many plant b-amylases
are spatially separated from their presumed substrate (i.e.
starch), and (c) inbred lines of rye lacking the abundant
endosperm b-amylase germinate normally [9]. This implies
that some b-amylases are not required and even not involved
in starch degradation but fulfil another role [10]. It has been
proposed, for example, that the abundant b-amylases from
cereal endosperm and alfalfa taproots function as seed
storage proteins and vegetative storage proteins (VSPs),

in general are transported from the cytoplasm into another
subcellular compartment.
A recent study of the predominant proteins in rhizomes of
hedge bindweed (Calystegia sepium ) revealed that this
vegetative storage tissue accumulates, besides large
quantities of a catalytically inactive RNase-related protein
[14], substantial amounts of a mannose/maltose-specific
lectin [15,16] and a 55-kDa polypeptide with an N-terminal
sequence similar to that of typical plant b-amylases. This is
an interesting observation because it demonstrates for the
first time the simultaneous occurrence in a plant tissue of a
lectin with a high affinity for the reaction product of
b-amylases. To confirm the possible interaction between the
carbohydrate-binding protein and the polysaccharide-
degrading enzyme the hedge bindweed b-amylase was
purified, characterized and immunolocalized. Our results
demonstrate that the enzyme resembles previously
described plant b-amylases and is exclusively located
in the cytoplasm. The abundance, subcellular location
and developmental regulation suggest that the rhizome
b-amylase is a cytoplasmic VSP.
MATERIALS AND METHODS
Plant material
Rhizomes of hedge bindweed [C. sepium (L.) R.Br.] were
collected in Leuven in winter.
Extraction and purification of b-amylase from rhizomes of
C. sepium
The b-amylase was purified by classical protein purification
techniques. Fresh rhizomes (100 g) were cut into small
pieces and homogenized in a Waring blender in 1 L of a

phenyl–Sepharose (Amersham Pharmacia Biotech,
Uppsala, Sweden) equilibrated with 1
M ammonium sulfate.
Bound proteins were eluted with 5 mL of 0.1
M Tris/HCl
(pH 8.7) and loaded onto a column (2.6 £ 70 cm;
< 350 mL bed volume) of Sephacryl 100 equilibrated
with KCl/NaCl/P
i
(1.5 mM KH
2
PO
4
/10 mM Na
2
HPO
4
/
3m
M KCl/140 mM NaCl, pH 7.4). The main peak eluting
with an apparent molecular mass around 200 kDa was
collected, dialysed against appropriate buffers and stored in
small aliquots at 2 20 8C until use. Analysis by SDS/PAGE
confirmed that the purified protein consisted exclusively of a
single 55-kDa polypeptide. Activity assays demonstrated
that the protein exhibited b-amylase activity.
Analytical methods
Purified proteins were analyzed by SDS/PAGE using
12.5–25% (w/v) acrylamide gradient gels as described by
Laemmli [17]. The gel was scanned with an AlphaImagere

M and the mixture kept on ice for 30 min The reaction
was quenched by the addition of 2-mercaptoethanol (50 m
M
final concentration) followed by heating at 60 8C for 10 min
Mass spectrometry was performed using a MALDI-TOF
instrument. Two microliters of a 1.3-mg·mL
21
b-amylase
solution were mixed with one microliter of a 50-m
M solution
of a-cyano-4-hydroxycinnamic acid in CH
3
CN/EtOH/
trifluoroacetic acid (50 : 49.9 : 0.1) and applied on the
multi sample target. This mixture was air-dried and the
target was then introduced in the instrument, a VG Tofspec
SE (Micromass, Manchester, UK) equipped with a N2-laser
(337 nm). The samples were measured in the linear mode
(acceleration voltage 25 kV), the laser energy was reduced
until an optimal resolution and signal/noise ratio was
obtained. The results of 10–20 shots were averaged to
obtain the final spectrum.
Enzyme assay
The b-amylase activity was determined by different
methods. The first method was based on the release of
6264 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001
p-nitrophenol from the specific substrate p-nitrophenyl
maltopentaoside (Betamyl reagent from Megazyme,
Wicklow, Ireland) [19]. Assays were performed for
10 min at 40 8C in maleate buffer (pH 6.2), and absorbance

incubation at 20 8C for 15 s to 2.5 min the reaction was
stopped by adding 0.5 mL 1
M HCl. To each sample 1 mL
staining solution (0.2% iodine in 2% potassium iodide) was
added, and the mixture diluted to 20 mL before measuring
the decrease in absorbance at 700 nm. The iodine staining
method also is not very specific for b-amylases and is
relatively insensitive. However, the method is less time
consuming than the dinitrosalicylic acid method and is not
affected by the inhibitors of the enzymatic activity.
Therefore the iodine staining method is well suited for
extensive kinetic analyses of purified b-amylases.
Stability tests
The heat stability of the enzyme was determined by heating
a solution of the purified protein (0.1 mg·mL
21
in 0.1 M
phosphate buffer pH 6.2) at 20 – 100 8C(with108C
increments) for 10 min. Afterwards, activity of the enzyme
was determined using the Betamyl b-amylase test reagent
(Megazyme, Wicklow, Ireland).
To determine the pH stability of the b-amylase, aliquots
of a solution of the purified protein (4.06 mg·mL
21
in water)
were adjusted to different pH values in a range between 2
and 12, and incubated for 1 h at 25 8C. Then 0.1 vol. of a
solution of 0.5
M sodium acetate (pH 5.0) was added and the
activity of the enzyme was measured by the dinitrosalicylic

were analysed using programs from
PC GENE (Intelli-
genetics, Mountain View, CA, USA) and
GENEPRO
(Riverside Scientific, Seattle, USA).
Northern blot analysis
RNA electrophoresis was performed according to Maniatis
et al. [25]. Approximately 3 mg of poly(A)-rich RNA were
denatured in glyoxal and dimethylsulfoxide and separated in
a 1.2% (w/v) agarose gel. Following electrophoresis the
RNAwastransferredtoImmobilonNmembranes
(Millipore, Bedford, USA) and the blot hybridized using a
random-primer-labeled cDNA insert or an oligonucleotide
probe. Hybridization was performed as reported by Van
Damme et al. [26]. An RNA ladder (0.16– 1.77 kb) was
used as a marker.
PCR amplification of genomic DNA fragments encoding
b-amylase
DNA was extracted from young leaves of C. sepium using
the protocol described by Stewart and Via [27]. The DNA
preparation was treated with RNase (Roche Diagnostics
GmbH, Mannheim, Germany). The reaction mixture for
amplification of genomic DNA sequences contained 10 m
M
Tris/HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl
2
, 100 mg·L
21
gelatin, 0.4 mM of each dNTP, 2.5 U of Taq polymerase
(Roche Molecular Biochemicals, Mannheim, Germany),

5 min) and processed immediately by affinity chromato-
graphy on a column of immobilized b-amylase or
CalsepRRP. Coupling of the antigens to the column and
purification of the antiserum were performed as described
previously [28].
Western blot analysis
The specificity of the antisera was analysed by Western blot
analysis. Proteins were separated by SDS/PAGE and
electroblotted on an Immobilon P membrane. Before
immunodetection the free binding sites on the membrane
were blocked with 5% BSA in Tris/NaCl/P
i
(10 mM Tris,
150 m
M NaCl, 0.1% Triton X-100, pH 7.6) for 1 h at room
temperature. After washing the membrane with Tris/NaCl/P
i
for 5 min the membrane was consecutively treated with
rabbit primary antibody (overnight incubation at room
temperature), goat anti-(rabbit IgG) Ig (1 h incubation at
room temperature) and peroxidase–anti-peroxidase com-
plex (1 h incubation at room temperature). After every
treatment the membrane was washed three times with
Tris/NaCl/P
i
for 5 min. Prior to the immunodetection the
membrane was washed for 5 min with 0.1
M Tris/HCl,
pH 7.6. The peroxidase reaction was carried out in a fresh
solution of 0.1

Axioskop epifluorescence microscope using the appropriate
filter combination. Micrographs were taken by a CCD
camera (Sony, Japan) and processed through the
PHOTOSHOP
program (Adobe, Seattle, WA, USA).
Molecular modelling
Multiple amino-acid sequence alignments based on
CLUSTAL W [30] were performed with SEQPUP (D.G. Gilbert,
Biology Department, Indiana University, Bloomington, IN,
USA). The program
SEQVU (J. Gardner, The Garvan
Institute of Medical Research, Sydney, Australia) was used
to compare the amino-acid sequences of the b-amylases.
Hydrophobic cluster analysis (HCA) [31,32] was
performed to delineate the structurally conserved b sheets
and a helices along the amino-acid sequences of the
b-amylase from hedge bindweed and the model b-amylase
from soybean. HCA plots were generated using the program
HCA-Plot2 (Doriane, Paris, France).
Molecular modeling of the b-amylase from C. sepium
was carried out on a Silicon Graphics O2 R10000
workstation, using the programs
INSIGHT II, HOMOLOGY
AND DISCOVER
(Molecular Simulations, San Diego CA,
USA). The atomic coordinates of the soybean b-amylase
(code 1BYA) [33] were taken from the RCSB Protein
Data Bank (http://www.rcsb.org/pdb) to build the three-
dimensional model of the C. sepium b-amylase. Energy
minimization and relaxation of the loop regions was carried

amylase were calculated using the
GRASP facilities.
RESULTS
Purification and partial characterization of the b-amylase
from
C. sepium
rhizomes
SDS/PAGE of clarified homogenates from resting hedge
bindweed rhizomes revealed several major polypeptides
(Fig. 1A), some of which have been identified previously.
The 15-kDa polypeptide (< 30% of the total protein)
corresponds to the subunits of the mannose-binding
C. sepium agglutinin (also called Calsepa) [15,16] whereas
the 26 to 28-kDa polypeptides (together < 35% of the total
protein) represent the unglycosylated and glycosylated form
of the so-called CalsepRRP [14]. N-terminal sequencing of
the 55-kDa polypeptide (< 10% of the total protein) yielded
6266 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001
a single sequence APIPGVMPMGNYVPVYVMLP with a
high degree of identity (85%) to the N-terminus of the
b-amylase from sweet potato (Ipomoea batatas ). Therefore
this polypeptide was tentatively identified as a b-amylase.
Subsequently the 55-kDa C. sepium protein was isolated
and tested for b-amylase activity.
The C. sepium b-amylase was purified using a combi-
nation of conventional protein purification techniques.
Analysis of a reduced sample of the final preparation by
SDS/PAGE yielded a single polypeptide band of < 55 kDa
(Fig. 1A). The unreduced protein also yielded a major band
of 55 kDa but exhibited an additional minor band of slightly

, 2.143 mg·mL
21
and 2.679 mg·mL
21
, respectively. The substrate concentration ranged
between 0.025 and 0.5% (w/v). (B) Lineweaver–Burk plots of the
inhibition of C. sepium b-amylase by glucose, maltose and cyclo-
hexaamylose. The activity was assayed using soluble starch as substrate
in 20 m
M, acetate buffer pH 5.0 at 20 8C. The concentration of enzyme
was 2.14 mg·mL
21
. Concentrations of glucose, maltose and cyclo-
hexaamylose were 80 m
M,80mM and 1.25 mM, respectively. The
substrate concentration ranged between 0.025 and 0.5% (w/v).
Fig. 1. SDS/PAGE and isoelectric focusing. (A) SDS/PAGE of a
clarified homogenate from C. sepium rhizomes and purified b-amylase.
Samples were loaded as follows: lane 1, 100 mL total extract from
Calystegia rhizomes; lanes 2–5, 20 mg purified b-amylase. The major
protein bands in crude extract (lane 1) represent the b-amylase (A), the
glycosylated and unglycosylated RNase-related protein (R) and
the lectin Calsepa (L). Protein samples in lanes 3 and 5 were alkylated.
The samples in lanes 1–3 were treated with b-mercaptoethanol; the
protein in lanes 4–5 was not reduced. Molecular mass reference
proteins (lane R) were lysozyme (14 kDa), soybean trypsin inhibitor
(20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine
serum albumin (67 kDa) and phosphorylase b (94 kDa). (B) Isoelectric
focusing of b-amylase from C. sepium rhizomes. Samples were loaded
as follows: Lane 1, purified b-amylase from C. sepium rhizomes and

125 m
M both sugars reduced the activity of the b-amylase
with 87.5%. Mannose caused a 6% reduction of the activity
when added at a final concentration of 125 m
M. In contrast,
lactose did not inhibit the enzyme even when the
concentration was increased to 250 m
M. The inhibition of
the enzyme activity by glucose, maltose and cyclohexa-
amylose was studied in more detail using the iodine staining
method. Glucose behaved as a mixed type inhibitor whereas
maltose and cyclohexaamylose behaved as competitive
inhibitors of the Calystegia b-amylase (Fig. 2B). Glucose
was only a weak inhibitor (K
i
¼ 262 mM) when compared
to maltose (K
i
¼ 11.7 mM) and cyclohexaamylose
(K
i
¼ 0.36 mM).
Molecular cloning of the
C. sepium
b-amylase
Screening of a cDNA library constructed with poly(A)-rich
RNA from rhizome apexes using a synthetic oligonucleotide
derived from the amino-acid sequence of the C. sepium
b-amylase yielded multiple positive clones of < 2Kb.
Sequence analysis of Calsepam1 revealed that this clone

dinucleotides at their 5
0
and 3
0
boundaries, respectively,
and were inserted between the third letter of one codon and
the first letter of the following codon.
Molecular modeling of the
C. sepium
b-amylase
The amino-acid sequence of the b-amylase from C. sepium
exhibits 67.5% identity and 76.0% similarity, respectively,
with soybean b-amylase (Fig. 3). As the HCA plots of both
proteins are very similar, the structurally conserved regions
(a helices and b sheets) are readily recognized (results not
shown). Due to these structural homologies, a fairly accurate
three-dimensional model could be built for the b-amylase
from C. sepium using the X-ray coordinates of the soybean
b-amylase (Fig. 4). According to the Ramachandran plot of
this model the f and c angles of most of the residues are in
the allowed regions of low energy, except for Arg421 (f,
888; c, 1318). It should be mentioned, however, that in the
soybean b-amylase also this residue is located in a
disallowed region. As shown in Fig. 4, the model of the
b-amylase from C. sepium comprises (a) a core built up of a
bundle of eight parallel b strands surrounded by eight a
helices and thus exhibits a typical (a/b)
8
barrel structure, (b)
a smaller globular region consisting of three long loops

endodermis and rhizodermis (Fig. 6). In cross-section of
cells of the pith and the cortex, the vacuole is the dominant
organelle. The cytoplasm is visible only as a thin layer
adjacent to the cell wall. As shown in Fig. 6, the b-amylase
Fig. 3. Amino-acid sequences. (A) Deduced
amino-acid sequence of the b-amylase from
C. sepium. As the methionine at position 2 is the
first amino acid the residue preceding this
methionine is shown in lower case. The sequence
corresponding to the N-terminal sequence of the
protein is underlined. The arrowheads indicate the
positions of the intron sequences. (B) Comparison
of the amino-acid sequences of b-amylase from
C. sepium (this work) with those from Glycine max
(GenBank accession No. BAA09462), A. thaliana
(accession no. BAA07842), Ipomoea batatas
(accession no. BAA02286), Triticum aestivum
(accession No. P93594), Zea mays (accession no.
P55005) and Hordeum vulgare (accession no.
BAA04815). Please note that the last 17-amino-
acid residues at the C-terminus of H. vulgare
b-amylase are not shown. Deletions are indicated
by dashes and identical residues are boxed.
q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6269
is confined to the cytoplasm (Figs 6B,D). No label for anti-
(b-amylase) IgG was detectable within the large vacuoles,
which appeared as a dark area in the centre of the cells. To
clearly distinguish the cytoplasmic location of the C. sepium
b-amylase from that of a noncytoplasmic protein, sections
were also immunolabeled with antibodies raised against the

also is restricted to the removal of the N-terminal
methionine whereas that of a phloem-specific b-amylase
from A. thaliana includes the removal of an N-terminal
tetrapeptide [8,41]. Cereal b-amylases also are not co- or
post-translationally modified [42]. However, the abundant
endosperm-specific cereal b-amylases are ‘activated and
released’ during germination by the proteolytic removal of a
C-terminal peptide of < 50 amino-acid residues [1,43].
The three-dimensional model of the C. sepium b-amylase
strongly resembles that of the soybean [33,39] and sweet
potato [38] b-amylases and shares the typical (a/b)
8
barrel
core which is common to all other b-amylases of different
Fig. 4. Three-dimensional model of Calystegia sepium b-amylase
showing the central bundle of eight strands of b sheet (pink
coloured arrows, numbered 1–8) surrounded by eight a-helices
(coloured green, numbered 1–8). The a helix in violet does not
participate in the core (b/a)
8
TIM-barrel structure. The three acidic
residues involved in the catalytic activity (Asp102, Glu187 and Glu381)
are represented in ball-and-stick. The conserved loop 97–104
(homologous to loop L3 of the soybean b-amylase), which allows the
active site to close is coloured cyan (H). N and C refer to the N- and
C-terminus of the b-amylase sequence. Cartoon was generated with
MOLSCRIPT [51], BOBSCRIPT [52] and RASTER3D [53].
Fig. 5. Molecular surface of the modelled C. sepium b-amylase
showing the surface area (black) occupied by the three acidic
residues Asp102 (red), Glu187 (blue) and Glu381 (green) located in

globular region. This conformational change is required to
close the active site of the enzyme [40,48] and allow the
reaction to take place. Once the reaction is finished a new
conformational change is required to bring the loop into the
open position for subsequent release of the reaction product.
Due to the importance of the conformational changes the
loop segment L3 is highly conserved in all b-amylases from
plants and microorganisms (e.g. 97Gly-Gly-Asn-Val-Gly-
Asp-Ala-Val104 of Calystegia b-amylase and 96Gly-Gly-
Asn-Val-Gly-Asp-Ile-Val103 of the soybean b-amylase).
Docking experiments with maltose and maltose derivatives
further suggested that the movement of this mobile flap
significantly increased the intermolecular binding potential
and thus favours the interaction with the ligand [49].
Immunolocalization studies of the C. sepium b-amylase
provided for the first time unequivocal evidence for the
exclusive cytoplasmic location of a plant b-amylase. Our
results confirm the presumed cytoplasmic location b-amy-
lases proposed on the basis of cell fractionation studies with
spinach [11] and Arabidopsis [7] leaves but can not be
reconciled with the previously proposed vacuolar location of
b-amylase in pea and wheat leaf protoplasts [12]. In contrast
to the cytoplasmic b-amylase, the major storage protein of
the hedge bindweed rhizome (CalsepRRP) [14] is clearly
located in the vacuole. This particular vacuolar location of
CalsepRRP not only serves as a good endogenous control
for the cytoplasmic location of the C. sepium b-amylase but
also demonstrates that cells of C. sepium rhizomes
accumulate large quantities of proteins in both the vacuole
and the cytoplasm.

q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6271
classified as a VSP (even though the C. sepium rhizome can
not be considered a true perennial tissue because it
continuously grows at the one end and dies at the other
end). If so, the C. sepium and alfalfa taproot b-amylases
represent a unique type of VSP because they are located in
the cytoplasm whereas all other known VSP are (presumed)
vacuolar storage proteins [50].
ACKNOWLEDGEMENTS
This work was supported in part by grants from the Research Fund
K.U.Leuven (OT/98/17), CNRS and the Conseil re
´
gional de Midi-
Pyre
´
ne
´
es (A. B., P. R.), and the Fund for Scientific Research-Flanders
(FWO grant G.0223.97). E. V. D. is a Postdoctoral Fellow of this fund.
REFERENCES
1. Ziegler, P. (1999) Cereal beta-amylases. J. Cereal Sci. 29,
195–204.
2. Mikami, B., Nomura, K. & Morita, Y. (1986) N-terminal sequence
of soybean b-amylase. J. Biochem. 100, 513–516.
3. Yoshida, N. & Nakamura, K. (1991) Molecular cloning and
expression in Escherichia coli of cDNA encoding the subunit of
sweet potato b-amylase. J. Biochem. 110, 196–201.
4. Gana, J.A., Kalengamaliro, N.E., Cunningham, S.M. & Volenec,
J.J. (1998) Expression of b-amylase from alfalfa taproots. Plant
Physiol. 118, 1495– 1505.

14. Van Damme, E.J.M., Hao, Q., Barre, A., Rouge
´
, P., Van Leuven, F.
& Peumans, W.J. (2000) Major protein of resting rhizomes of
Calystegia sepium (hedge bindweed) closely resembles plant
RNases but has no enzymatic activity. Plant Physiol. 122,
433–445.
15. Van Damme, E.J.M., Barre, A., Verhaert, P., Rouge
´
, P. & Peumans,
W.J. (1996) Molecular cloning of the mitogenic mannose/maltose-
specific rhizome lectin from Calystegia sepium. FEBS Lett. 397,
352–356.
16. Peumans, W.J., Winter, H.C., Bemer, V., Van Leuven, F., Goldstein,
I.J., Truffa-Bachi, P. & Van Damme, E.J.M. (1997) Isolation of a
novel plant lectin with an unusual specificity from Calystegia
sepium. Glycoconjugate J. 14, 259–265.
17. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T
4
. Nature 227, 680 –685.
18. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F.
(1956) Colorimetric method for determination of sugar and related
substances. Anal. Chem. 28, 350–356.
19. Mathewson, P.R. & Seabourn, B.W. (1983) A new procedure for
specific determination of b-amylase in cereals. J. Agric. Food
Chem. 31, 1322– 1326.
20. Bernfeld, P. (1955) Amylases, a and b. Methods Enzymol. 1,
149–158.
21. Nomura, K., Mikami, B. & Morita, Y. (1986) Interaction of

alignment through sequence weighting, position specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22,
4673–4680.
31. Gaboriaud, C., Bissery, V., Benchetrit, T. & Mornon, J.P. (1987)
Hydrophobic cluster analysis: an efficient new way to compare and
analyse amino acid sequences. FEBS Lett. 224, 149–155.
32. Lemesle-Varloot, L., Henrissat, B., Gaboriaud, C., Bissery, V.,
Morgat, A. & Mornon, J.P. (1990) Hydrophobic cluster analysis:
procedure to derive structural and functional information from
2-
D-representation of protein sequences. Biochimie 72, 555–574.
33. Mikami, B., Dagano, M., Hehre, E.J. & Sacchettini, J.C. (1994)
Crystal structure of soybean b-amylase reacted with b-maltose and
maltal: active site components and their apparent roles in catalysis.
Biochemistry 33, 7779–7787.
34. Ponder, J.W. & Richards, F.M. (1987) Tertiary templates for
proteins. Use of packing criteria in the enumeration of allowed
sequences for different structural classes. J. Mol. Biol. 193,
775–791.
35. Mas, M.T., Smith, K.C., Yarmush, D.L., Aisaka, K. & Fine, R.M.
(1992) Modeling the anti-CEA antibody combining site by
homology and conformational search. Proteins 14, 483–498.
36. Nicholls, A., Sharp, K.A. & Honig, B. (1991) Protein folding and
association: insights from the interfacial and thermodynamic
properties of hydrocarbons. Proteins 11, 281–296.
6272 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001
37. Gilson, M.K. & Honing, B.H. (1987) Calculation of electrostatic
potential in an enzyme active site. Nature 330, 84–86.
38. Cheong, C.G., Eom, S.H., Chang, C., Shin, D.H., Song, H.K., Min,
K., Moon, J.H., Kim, K.K., Hwang, K.Y. & Suh, S.W. (1995)

46. Totsuka, A. & Fukazawa, C. (1996) Functional analysis of Glu380
and Leu383 of soybean beta-amylase. A proposed action
mechanism. Eur. J. Biochem. 240, 655–659.
47. Totsuka, A., Nong, V.H., Kadokawa, H., Kim, C.S., Itoh, Y. &
Fukazawa, C. (1994) Residues essential for catalytic activity of
soybean beta-amylase. Eur. J. Biochem. 221, 649–654.
48. Kunikata, T., Nishimura, S. & Nitta, Y. (1996) Maltal binding
mechanism and a role of the mobile loop of soybean beta-amylase.
Biosci. Biotechnol. Biochem. 60, 1104–1108.
49. Laederach, A., Dowd, M.K., Coutinho, P.M. & Reilly, P.J. (1999)
Automated docking of maltose, 2-deoxymaltose, and maltotetraose
into the soybean b-amylase active site. Proteins 37, 166–175.
50. Staswick, P.E. (1994) Storage proteins of vegetative plant tissues.
Annu. Rev. Plant Physiol. Mol. Biol. 45, 303–322.
51. Kraulis, P.J. (1991) Molscript: a program to produce both detailed
and schematic plots of protein structures. J. Appl. Crystallogr. 24,
946–950.
52. Esnouf, R.M. (1997) An extensively modified version of Molscript
that includes greatly enhanced coloring capabilities. J. Mol.
Graphics 15, 132–134.
53. Merritt, E.A. & Bacon, D.J. (1997) Raster3D photorealistic
molecular graphics. Methods Enzymol. 277, 505 –524.
q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6273