The ‘pair of sugar tongs’ site on the non-catalytic domain C
of barley a -amylase participates in substrate binding and
activity
Sophie Bozonnet
1,2
, Morten T. Jensen
2
, Morten M. Nielsen
1
, Nushin Aghajari
3
, Malene H. Jensen
3
,
Birte Kramhøft
1,2
, Martin Willemoe
¨
s
1,2
, Samuel Tranier
3
, Richard Haser
3
and Birte Svensson
1,2
1 Enzyme and Protein Chemistry, BioCentrum-DTU, Technical University of Denmark, Kgs. Lyngby, Denmark
2 Carlsberg Laboratory, Valby, Denmark
3 Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Prote
´
ines, Universite

This site on the non-catalytic domain C in the C-terminal part of the mole-
cule contains a key residue, Tyr380, which has numerous contacts with the
oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to
b-cyclodextrin-Sepharose, a starch-mimic resin used for a-amylase affinity
purification. The K
d
for b-cyclodextrin binding to Y380A and Y380M was
1.4 mm compared to 0.20–0.25 mm for the wild-type, S378P and S378T
enzymes. The substitution in the S378P enzyme mimics Pro376 in the bar-
ley a-amylase 2 isozyme, which in spite of its conserved Tyr378 did not
bind oligosaccharide at the ‘sugar tongs’ in the structure. Crystal structures
of both wild-type and S378P enzymes, but not the Y380A enzyme, showed
binding of the pseudotetrasaccharide acarbose at the ‘sugar tongs’ site. The
‘sugar tongs’ site also contributed importantly to the adsorption to starch
granules, as K
d
¼ 0.47 mgÆmL
)1
for the wild-type enzyme increased to
5.9 mgÆmL
)1
for Y380A, which moreover catalyzed the release of soluble
oligosaccharides from starch granules with only 10% of the wild-type activ-
ity. b-cyclodextrin both inhibited binding to and suppressed activity on
starch granules for wild-type and S378P enzymes, but did not affect these
properties of Y380A, reflecting the functional role of Tyr380. In addition,
the Y380A enzyme hydrolyzed amylose with reduced multiple attack,
emphasizing that the ‘sugar tongs’ participates in multivalent binding of
polysaccharide substrates.
Abbreviations

on stability
and activity [5,13] and with the AMY2-specific sensi-
tivity to barley a-amylase ⁄ subtilisin inhibitor (BASI)
[5,14,15]. AMY1 furthermore binds substrates – starch
granules included – more tightly than does AMY2,
which shows a higher turn-over rate than AMY1
[16–18]. Domain-C is present in almost all GH-H
members and its functional role has not yet been
assigned. Remarkably, the ‘sugar tongs’ site defined
around Tyr380 in domain-C of AMY1 and binding
malto-oligosaccharide [4] was not occupied in the
structure of AMY2 [3] although this critical tyrosine is
conserved in AMY2.
AMY1, AMY2, and other GH-H enzymes possess
different secondary carbohydrate-binding sites that are
not part of the active site area but which are situated
on the surface of the catalytic domain or an inti-
mately associated domain rather than on a CBM, e.g.
a starch-binding domain (SBD) [1,3,4,19–22]. The role
of multivalent binding in enzymatic degradation of
polysaccharides is in general not clearly understood at
the molecular level. In amylolytic enzymes such sites
are thought to (a) ensure association with starch gran-
ules, (b) assist in disentangling of a-glucan chains,
(c) guide the substrate chain to the active site, and
(d) confer allosteric regulation. Multivalent binding is
also envisaged in the multiple attack mechanism
proposed in the late 1960s for amylose degradation
by a-amylase, in which an initial endo-attack was
followed by hydrolysis of more glucosidic bonds

the indole side chains of Trp278Trp279 on the surface
Fig. 1. Close-up view on the ‘pair of sugar tongs’ binding site in
the crystal structure of a-amylase 1 (AMY1) D180A, an inactive cat-
alytic nucleophile mutant, in complex with maltoheptaose [21].
Important residues defining this site have been highlighted. Ser378
and Tyr380 are mutated in the present work. As continuous elec-
tron density was only found for five sugar rings, a pentasaccharide
was modeled into the structure.
a-amylase ‘sugar tongs’ mutants S. Bozonnet et al.
5056 FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS
of domain A [21]. Seven rings in a third maltoheptaose
molecule occupied subsites )7 through )1 in the active
site [21]. Noticeably, AMY2 accommodated the
pseudotetrasaccharide inhibitor acarbose both at
Trp276Trp277 and at the active site, but not at the
‘sugar tongs’ [3]. Comparison of AMY1 and AMY2
structures [3,4] suggested that Pro376AMY2 – corre-
sponding to Ser378AMY1 – rigidified the loop carry-
ing Tyr378
AMY2
(Tyr380 in AMY1), hindering the
conformational shift needed in oligosaccharide binding
[4]. Different secondary carbohydrate-binding sites are
found in GH-H members, e.g. certain a-amylases
[3,4,22,26–28], cyclodextrin glucosyltransferase [29],
amylosucrase [30], amylomaltase [20], and Thermoacti-
nomyces vulgaris I amylase [31]. The Pseudomonas
maltotetraose-forming amylase structure closely resem-
bles that of AMY1 but has no tyrosine at the position
of Tyr380 [4]. Tyr380, however, is present in several

proline; S378T represented rice and millet a-amylases
[33] (Fig. 2). The host Pichia pastoris secreted
10–44 mgÆL
)1
wild-type and AMY1 mutants as esti-
mated from specific activities against insoluble
blue starch (iBS) of the purified enzymes (Table 1).
Fig. 2. Sequence alignment of domain-C of barley AMY1 and AMY2, four other cereal amylases, and a legume a-amylase. The secondary
structure of AMY1 is indicated above the alignment and mutated residues are highlighted in orange. Accession numbers are; wheat (AMY3):
P08117; maize: Q41770; millet: Q7Y1C3; rice (AMY3): P27933; kidney bean: Q9ZP43.
Table 1. Enzymatic properties of ‘sugar tongs’ mutants of barley a-amylase 1 (AMY1). U, one enzyme unit is the amount required to cause
an A
620
increase of 1.
Enzyme
iBS Amylose DP440 Cl-pNPG
7
Specific activity
(UÆmg
)1
)
k
cat
(s
)1
)
k
m
(mgÆmL
)1

Y380M 2000 149 ± 44 0.351 ± 0.083 424.5 34 ± 0.8 0.871 ± 0.027 39.0
Y380F 2790 162 ± 27 0.391 ± 0.146 414.3 56 ± 1.7 0.724 ± 0.123 77.3
S378P 2695 163 ± 36 0.203 ± 0.130 802.9 59 ± 0.6 0.861 ± 0.023 68.5
S378T 2705 144 ± 9 0.208 ± 0.058 692.3 48 ± 1.7 0.735 ± 0.087 65.3
AMY1 2500 185 ± 20 0.190 ± 0.010 973.7 52 ± 4.9 0.758 ± 0.112 68.6
AMY2 4000 721 ± 63 1.074 ± 0.283 671.3 86 ± 3.1 2.125 ± 0.180 40.5
S. Bozonnet et al. a-amylase ‘sugar tongs’ mutants
FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS 5057
Similarly to the AMY1 wild-type, S378P, S378T and
Y380F were obtained in $50% yield by affinity chro-
matography on b-cyclodextrin (b-CD)-Sepharose,
whereas Y380A and Y380M AMY1 did not bind to
the resin and were purified in $20% yield by ammo-
nium sulfate precipitation and ion exchange chroma-
tography (see Experimental procedures).
Enzymatic activity of ‘sugar tongs’ AMY1
mutants
Replacement of Tyr380 by alanine and methionine
caused 50–75% reduction in the activity of iBS (k
cat
),
amylose DP440 (k
cat
⁄ K
m
), and even the oligosaccha-
ride Cl-pNPG
7
(k
cat

This property was lost in Y380A, suppressed for
Y380M, but retained by Y380F, S378P, and S378T
AMYl, and was missing for AMY2 (data not shown).
The earlier reported hydrolysis of the amylose of
DP440 in a multiple attack mechanism [12] was con-
firmed for AMY1, which showed a DMA of 1.9 as
determined from the ratio of rates of release of
reducing groups in the fraction of small (i.e. ethanol-
soluble) products over large (i.e. ethanol-precipitated)
products (see Experimental procedures and [12]). The
rates of product formation by the mutants (not
shown) agreed with the activity levels described in
Table 1. AMY1 Y380A had a DMA of 1.0 and thus
released fewer short products per enzyme–substrate
encounter than AMY1 wild-type, whereas AMY1
Y380M and S378P maintained a DMA of 2.0 and
2.2, respectively.
Binding of b-cyclodextrin to ‘sugar tongs’
mutants measured by surface plasmon resonance
analysis
Surface plasmon resonance (SPR) analysis was suitable
for measuring the affinity in the low millimolar range
of b-CD for AMY1. SPR sensorgrams clearly illustrated
weaker binding to AMY1 Y380A than to wild-type
enzyme (Fig. 3) and K
d
was calculated to 1.40 mm for
both Y380A and Y380M, i.e. sevenfold higher than K
d
of AMY1 wild-type (Table 2). Y380F caused only a

Fig. 3. b-CD binding determined by SPR analysis. AMY1: d wild-
type, s Y380A. Response unit (RU) values are corrected for the
contribution given by a channel in the chip without bound enzyme
protein.
Table 2. Binding of b-cyclodextrin (b-CD) to ‘sugar tongs’ mutants
and wild-type AMY1 and AMY2 as determined by SPR. See Experi-
mental procedures for the SPR analytical procedure.
Enzyme K
d
(mM)
Y380A 1.40 ± 0.23
Y380M 1.39 ± 0.65
Y380F 0.36 ± 0.02
S378P 0.25 ± 0.03
S378T 0.23 ± 0.02
AMY1 0.20 ± 0.04
AMY2 0.63 ± 0.27
a-amylase ‘sugar tongs’ mutants S. Bozonnet et al.
5058 FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS
very important role of Tyr380 is in accordance with
b-CD having no impact on the apparent affinity of
AMY1 Y380A for starch granules, whereas the presence
of 0.5 mm b-CD increased the apparent K
d
four- to six-
fold for AMY1 wild-type and S378P and AMY2
(Table 3), confirming competition in binding to starch
granules. AMY2 showed threefold weaker affinity for
barley starch granules than did both the wild-type and
the AMY2 mimic, AMY1 S378P (Table 3).

(Table 1). The activity was reduced in the presence of
b-CD (Fig. 4; Table 4) due to competition with starch
granule binding. The low activity hampered analysis of
the effect of b-CD on AMY1 Y380A.
Table 3. Binding of ‘sugar tongs’ mutants, wild-type AMY1 and
AMY2 to barley starch granules. The binding was measured in the
range 0.01–40 mgÆmL
)1
starch granules (see Experimental proce-
dures and [29] for details). (A) no b-CD; (B) in the presence of
0.5 m
M b-CD.
Enzyme K
d
(mgÆmL
)1
)B
max
A
Y380A 5.90 ± 0.47 0.90 ± 0.05
S378P 0.57 ± 0.04 0.98 ± 0.01
AMY1 0.47 ± 0.06 1.03 ± 0.04
AMY2 1.27 ± 0.32 0.99 ± 0.03
B
Y380A 6.86 ± 0.55 0.81 ± 0.02
S378P 2.93 ± 0.36 0.95 ± 0.02
AMY1 2.85 ± 0.28 0.98 ± 0.02
AMY2 4.63 ± 0.37 0.87 ± 0.02
Fig. 4. Rates of release of soluble reducing products from barley starch granules as catalyzed by AMY1, AMY2, and Y380A and
S378P AMY1 in the absence (d), and in the presence (s) of 0.5 m

(molecule A) the electron density for rings B and C
was very clear (Fig. 6A) and almost entirely defined
for ring A; however, the density was badly defined for
ring D, which therefore was not inserted for refine-
ment. In molecule B of S378P ⁄ acarbose, rings B and C
were completely defined, ring D was better defined
than molecule A, and ring A was poorly defined. In
molecule C, rings A–C were defined very clearly,
whereas ring D lacked continuous electron density and
was omitted from the refinement. In spite of cocrystal-
lization, a hydrated calcium ion (Ca503) and not
acarbose was bound at the active site of AMY1 S378P
(not shown). Ca503 was also present in native S378P
(not shown) and it was previously observed in
Table 4. Hydrolysis of barley starch granules by ‘sugar tongs’
mutants, wild-type AMY1 and AMY2. (A) no b-CD; (B) in the pres-
ence of 0.5 m
M b-CD. NC, not calculated due poor affinity; ND, not
determined due to low activity. See also Fig. 4. See Experimental
procedures for details on the procedure.
A
Enzyme k
cat
(s
)1
) K
m
(mgÆmL
)1
)

loop region, Ala211–Pro218.
Fig. 6. Close-up view on the ‘sugar tongs’ binding site. (A)
S378P ⁄ acarbose (molecule A), showing the bound sugar ligand
(rings A, B, and C) and (B) Y380A ⁄ acarbose, which has no ligand
bound at the ‘sugar tongs’ site.
a-amylase ‘sugar tongs’ mutants S. Bozonnet et al.
5060 FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS
AMY1Æthio-DP4 [4] as well as in AMY2ÆBASI (a pro-
teinaceous inhibitor complex) [37], but so far not in
native AMY1.
At the surface-binding site containing Trp278Trp279
on the (b ⁄ a)
8
-barrel [3,4,21], electron density studies
identified three sugar rings in AMY1 S378P ⁄ acarbose,
and in wild-type AMY1 ⁄ acarbose sugar binding
occurred at this site as well as to the active site cleft
and the ‘sugar tongs’ [21]. The three rings defined at
the Trp278Trp279 site corresponded to acarbose with
the reducing-end glucose cleaved off and with the same
orientation, but shifting position by one sugar unit
compared to the ligand in S378P ⁄ acarbose (mole-
cule A). Thus acarbose rings A and B stacked onto
Trp279 and Trp278, respectively, whereas ring C was
in the bulk solvent (not shown). In wild-type
AMY1 ⁄ acarbose, rings B and C stacked onto
Trp279Trp278. As a curiosity, rings A and B modeled
into the electron density on this surface site in
AMY2 ⁄ acarbose [3] were at the same position as in
the AMY2 mimic, S378P AMY1 ⁄ acarbose.

a molecular model in emulating protein–starch interac-
tions. Despite lack of binding to b-CD-Sepharose, the
SPR procedure developed in the present work enabled
analysis of b-CD affinity in the millimolar range for
AMY1 Y380A and Y380M. The K
d
of 1.40 mm for
b-CD was increased sevenfold, confirming the critical
functional role of Tyr380 in the ‘sugar tongs’. As
b-CD-Sepharose did not retain these mutants, their
still intact other surface site containing Trp278Trp279
was concluded to have very low affinity for b-CD.
b-CD accordingly was seen to bind at the ‘sugar
tongs’, and not at Trp278Trp279 in the structure of
AMY1 active site mutants (Tranier, Aghajari, Haser,
Mori and Svensson, unpublished). Crystallography on
AMY1 Y380A (present work) demonstrated that this
substitution destroyed acarbose binding to the ‘sugar
tongs’, whereas acarbose bound to Trp278Trp279.
Furthermore, acarbose occupied the ‘sugar tongs’ in
S378P ⁄ acarbose (the AMY2 mimic). Hence as
AMY1 S378P and wild-type also shared the same
affinity for b-CD, several properties of AMY1 S378P
did not confirm the earlier suggestion that Pro376
(AMY2-numbering) caused the lack of ligand binding
at the ‘sugar tongs’ in the AMY2 structure [3,4]. The
modest threefold weaker affinity seen for b-CD bind-
ing by AMY2 compared to AMY1 wild-type and
S378P, possibly combined with different crystallization
conditions for AMY1 and AMY2 [3,50,51], may have

granules (K
d
¼ 7.6 mgÆmL
)1
), and its K
d
increased
S. Bozonnet et al. a-amylase ‘sugar tongs’ mutants
FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS 5061
only two- to threefold for SBD single and dual binding
site mutants [29]. The homologous SBD of Aspergillus
niger glucoamylase had K
d
values of 6.4 and 28 lm for
b-CD and of 0.95 and 17 lm for maltoheptaose for
each of the two binding sites, respectively [40]. Thus
AMY1 ‘sugar tongs’ and these SBDs show very differ-
ent ligand specificity, AMY1 having about 15-fold
higher and 30-fold lower affinity for starch granules
and b-CD, respectively, than does SBD. This is also
reflected in the lower B
max
values found for the cyclo-
dextrin glucosyltransferase [29].
AMY1 Y380A had only 10% hydrolytic activity of
wild-type against starch granules apparently due to
poor substrate binding. Furthermore, although b-CD
did not inhibit AMY1-catalyzed hydrolysis of amylose
[43], b-CD reduced the catalytic efficiency (k
cat

7
that covers only
seven to eight active site subsites [44]. This latter loss
in activity was speculated to stem from Cl-pNPG
7
binding to the ‘sugar tongs’, similarly to other oligo-
saccharides [21]. This binding may modulate activity,
as supported by the very detailed study of acarbose
inhibition kinetics of hydrolysis of amylose by barley
a-amylase, where acarbose was concluded to occupy at
least one secondary site in the productive enzyme–sub-
strate complex and, furthermore, that this binding al-
losterically enhanced activity [46]. As orientation of
maltoheptaose molecules bound to AMY1 D180A sug-
gested that three different, rather than the same, a-glu-
can chains were accommodated at the active site and
at the two surface sites [21], one cannot on a structural
basis, model interactions in the multiple attack mecha-
nism showing the substrate chain attached at the
‘sugar tongs’. Thus even though increased DMA of
the AMY1–SBD fusion suggested that enzyme–sub-
strate interactions at secondary binding sites were
favoring multiple attack [12,25], in agreement with the
reduced DMA of the AMY1 Y380A ‘sugar tongs’
mutant, these effects may stem from allosteric regula-
tion.
The mutational analysis of the ‘sugar tongs’ in barley
AMY1 explored the role of this so far unique carbohy-
drate-binding surface site from plant a-amylases. This
is the first demonstration of a function for a C domain

amplified using 5¢-TTTGAATTCCATGGGGAAGAACG
GCAGC-3¢ as sense orientation primer and a purified mega-
primer. Pfu DNA polymerase (Stratagene, La Jolla, CA) was
used for PCR and products were cut by NarI and KpnI. The
700 bp fragments were purified (QIAquick gel extraction kit,
QIAGEN, Germantown, MD) and subcloned in NarI, KpnI-
linearized pPICZA-amy1D9. Plasmids were propagated in
E. coli DH5a [low-salt LB, 25 lgÆmL
)1
Zeocin
Ò
(Invitrogen,
Carlsbad, CA)], purified (Midiprep Plasmid extraction kit,
QIAGEN), sequenced (Big-Dye premix; ABI PRISM 310
Genetic Analyzer, Perkin Elmer Life Sciences, Waltham,
MA), and BglII-linearized prior to transformation of P. pas-
toris by electroporation [48]. Transformants were identified
on YPDS (1% yeast extract, 2% peptone, 2% glucose, 1 m
sorbitol, 2% agar, 100 lgÆmL
)1
Zeocin), transferred to meth-
anol ⁄ starch plates and selected for a-amylase secretion by
halos seen by exposure to I
2
[10].
a-amylase ‘sugar tongs’ mutants S. Bozonnet et al.
5062 FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS
Enzyme production and purification
Pichia pastoris transformants were grown in 1 L BMGY
(1% yeast extract, 2% peptone, 1% glycerol, 0.67% yeast

min
)1
using buffer without and with 0.5 m NaCl (A
¨
KTA-
explorer, GE Healthcare). Two forms of differing pI were
resolved by anion exchange chromatography [10]. The first-
eluting and highly active form was dialyzed (10 mm Mes,
25 mm CaCl
2
, pH 6.8) and concentrated (Centriprep YM10,
Millipore), 0.02% (w ⁄ v) NaN
3
was added, and the form kept
at 4 °C, whereas the more acidic form containing gluta-
thionylated Cys95 [49] was discarded. All steps were carried
out at 4 °C. Proteins migrated as single bands in SDS ⁄ PAGE
and showed pI ¼ 4.8 by isoelectric focusing [9].
Enzyme activity
Insoluble blue starch
Enzyme was added (50 lL, final 1–12 nm) to 5 mg iBS
(Amersham Biosciences) in 20 mm Na acetate pH 5.5,
5mm CaCl
2
, 0.005% BSA (0.8 mL) and incubated at
37 °C. At 15 min, 0.5 m NaOH (200 lL) was added and
after centrifugation (10 000 g, 3 min) the absorbance of the
supernatants (300 lL, in duplicate) was measured at
620 nm in a microtiter plate reader (MRX-TC Revelation;
Dynex Technologies, Richfield, MN). One enzyme unit is

50 mm KCl, 0.02% NaN
3
, 3167 nkatÆmL
)1
Saccharomyces
cerevisiae a-glucosidase, and 50 nkatÆmL
)1
almond b-gluco-
sidase (both Sigma) were measured at 405 nm in microtiter
plates using 4-nitrophenol as standard. k
cat
and K
m
were
obtained as above.
Starch granules
Enzyme (final concentration 4–7 nm) was added to barley
starch granules (Primalco, Helsinki, Finland) at 10 concen-
trations (0.8–400 mgÆmL
)1
)in20mm Na acetate, pH 5.5,
5mm CaCl
2
, 0.005% BSA (w ⁄ v) agitated (1000 r.p.m.) at
37 °C. Hydrolysis was measured over 25 min as reducing
power in supernatants of centrifuged (10 000 g, 5 min,
room temperature) aliquots. k
cat
and K
m

calculated from at least triplicate experiments.
Surface plasmon resonance
Enzyme (0.9–1.1 nmol in 30–100 lL) was biotinylated and
immobilized on a streptavidin-coated chip, using BIAcore
S. Bozonnet et al. a-amylase ‘sugar tongs’ mutants
FEBS Journal 274 (2007) 5055–5067 ª 2007 The Authors Journal compilation ª 2007 FEBS 5063
3000 (BIAcore AB, Uppsala, Sweden) at $5ngÆmL
)1
in
running buffer [10 mm Mes, pH 6.5, 5 mm CaCl
2
, 0.005%
(v ⁄ v) surfactant P20] for 4 min at 10 lLÆmin
)1
[15] to reach
2000–3000 response units (RU). Sensorgrams (RU versus
time) were recorded of b-CD (12 concentrations,
15 lm)7mm) binding in running buffer at 30 lLÆmin
)1
and 25 °C for 3 min, followed by 3 min dissociation in
buffer. RU for a parallel flow cell without enzyme was
subtracted and K
d
was obtained by steady state affinity
fitting analysis (biaevaluation 3.1 software). Experiments
were carried out in triplicate.
Binding to starch granules
Enzyme (final 4–12 nm) was agitated 30 min with starch
granules at 10–13 concentrations (0.01–40 mgÆmL
)1

(Y380A ⁄ acarbose) and 0.3 · 0.02 · 0.01 mm
3
(S378P ⁄ acar-
bose), were cryo-protected by soaking a few seconds in
mother liquor made up to 10% (w ⁄ v) in ethylene glycol
and, for Y380A ⁄ acarbose, also 10 mm in acarbose. Data
were collected at beamline ID14-4 (European Synchrotron
Radiation Facility, Grenoble, France). Diffracted intensities
were integrated and scaled (xds program package [52]).
Crystal parameters and data collection statistics are given
in supplementary Table S1.
Structure determination and refinement
The S378P ⁄ acarbose structure was solved by molecular
replacement with AMY1 at 1.5 A
˚
resolution (Protein Data
Bank entry 1HT6) as search model [4], omitting water mole-
cules and calcium ions, and using data in the resolution range
15–3.5 A
˚
(cns software [53]). Initial rigid body refinement
included data to 3.5 A
˚
resolution; in the remaining refine-
ments a simulated annealing protocol was used extending
data up to 1.7 A
˚
combined with anisotropic B-factor refine-
ment. Due to crystal isomorphism with Y380A, wild-type
AMY1 [4] was used as the starting model in a difference Fou-

are summarized in supplementary Table S1.
Sequence alignment
Domain-C sequences from selected a-amylases were aligned
using clustalw [58]. Superimposition of secondary struc-
tures of AMY1 and rendering was done with the program
espript [59].
Acknowledgements
Sidsel Ehlers, Mette Hersom Bien, Lone Sørensen
(Carlsberg Laboratory) and Susanne Blume (Enzyme
and Protein Chemistry, BioCentrum-DTU) are grate-
fully acknowledged for excellent technical assistance,
and Peter K. Nielsen and Phaedria St. Hilaire for
advice on SPR analysis. Xavier Robert and Maher
Abou Hachem are thanked for stimulating discussions.
This work was supported by the European Union
Fourth Framework Program on Biotechnology (CT98-
0022, AGADE) and Fifth Framework Program ‘Qual-
ity of Life and Management of Living Resources’
(QLK3-2001–00149, CEGLYC), the Danish Natural
Science Research Council, the Carlsberg Foundation,
and a Ph.D. stipend from DTU (to MMN).
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Supplementary material