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9875–9884, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
The crystal structure of the ␣-amylase from the hyper- sequences of the other two domains, B and C, are void of any
thermophilic archaeon Pyrococcus woesei was solved in conserved motifs and are not involved directly in substrate
the presence of three inhibitors: acarbose, Tris, and catalysis.
zinc. In the absence of exogenous metals, this ␣-amylase Besides the recently solved three-dimensional structure of
bound 1 and 4 molar eq of zinc and calcium, respec- the glycosyltrehalose trehalosidase from Sulfolobus solfatari-
tively. The structure reveals a novel, activating, two- cus Km1 that exhibits ␣-amylase activity (8), no other struc-
metal (Ca,Zn)-binding site and a second inhibitory zinc- tures of archael class-13 ␣-amylases are known to date. The
binding site that is found in the ⴚ1 sugar-binding pocket ␣-amylase from the hyperthermophilic archaeon Pyrococcus
similar (root mean square deviation of 2.3 Å based on the C␣ crystal forms grown in the presence of zinc (PWA䡠Zn and
backbone) (27) to the corresponding domain of PWA. In con- PWA䡠Ac/Zn) reveal two additional metal sites, bringing the
trast, domain B of BLA contains 43 additional residues that total in these structures to seven. The presence of an anoma-
form a second -sheet (27). The C-terminal domain C (residues lous signal under the experimental conditions of x-ray data
341– 435) is arranged in an eight-stranded antiparallel -sheet collection was used to indicate the presence of zinc (Tables I
containing a Greek key motif. The function of this domain still and II). In the PWA䡠Tris structure, solved from crystals grown
remains unclear. in the absence of zinc, but in the presence of magnesium, only
We initially identified the nature of the bound metal ions by one site showed a significant anomalous signal and therefore
PIXE. For these experiments, PWA was expressed heterolo- was refined as a zinc site (Table II). The remaining four sites,
gously in E. coli and purified in the absence of exogenous without an anomalous signal, but retaining strong positive
metals except for sodium. The PIXE data revealed the presence difference electron density, when refined as an ordered solvent
of 1.1 ⫾ 0.4 molar eq of zinc and 4 ⫾ 2 molar eq of calcium (⬎10 ), were interpreted to be calcium or magnesium sites.
bound to PWA. In the PWA crystal structures, the type of the Thus, the stoichiometry of calcium (or magnesium) and zinc
metal sites was characterized by the analysis of anomalous sites observed in the PWA䡠Tris structure is in good agreement
x-ray data and positive peaks in Fo ⫺ Fc difference Fourier with the PIXE data. In contrast, in the PWA䡠Zn and
electron density maps (Table I). Based on these data, the PWA PWA䡠Ac/Zn structures, all sites except one showed a significant
structures show excessive metal binding; all three structures anomalous signal and were therefore refined as zinc sites. The
have five metal-binding sites in common (Table II). The two remaining site was interpreted as a calcium site. These metal
9878 Structure of a (Ca,Zn) Metal Center-containing ␣-Amylase
FIG. 2. Structure-based alignment of PWA sequences. The secondary structure assignments for PWA are shown as colored cylinders Downloaded from www.jbc.org by guest, on November 14, 2010
(310-helices and ␣-helices) and arrows (-strands) above the aligned sequences. AVA, Hordeum vulgare ␣-amylase (AMY2) chain A (Protein Data
Bank code 1AVA) (46). The Protein Data Bank code for BLA is 1BLI (27). The positions of amino acid residues coordinating the zinc ion in domain
B (site 1; cf. Table II) are highlighted in red; those coordinating calcium (site 2; cf. Table II) in domain B are colored yellow; and the cysteines
forming disulfide bonds are colored green. The seven invariant amino acid residues are marked in black. The alignment was carried out with DALI
using the main chain positions of each coordinate set.
sites will be referred to as activating sites (Table II, sites 1 and residual PWA activity was ⬍10%. In contrast to many other
2), an inhibiting site (site 7), and other sites (sites 3– 6). characterized ␣-amylases, including commercially available
Chemically Unrelated Inhibitors Competitively Bind to the BLA (10, 13), PWA was not activated significantly by an excess
Active Site—We quantified the inhibitory effects of a number of of calcium (Table III). Based on the measured inhibition data,
established ␣-amylase active site ligands, including the transi- we selected three ligands (acarbose, Tris, and zinc) for crystal-
tion state analog acarbose and the buffer Tris (Table III). To lographic characterization of the PWA active site (Fig. 3).
link the observed tight binding of calcium and zinc to a poten- Acarbose is a pseudotetrasaccharide inhibitor consisting of a
tial role in activity regulation, we also assessed the regulatory valienamine unit at the nonreducing end linked to 4-amino-
properties of a number of metal ions (Table III). Except for 4,6-dideoxy-␣-D-glucose, which is fused to maltose. In the
copper (full inhibition in the presence of 3 mM Cu2⫹), zinc had PWA䡠Ac/Zn structure, resulting from co-crystallization of PWA
the strongest inhibitory effect. In the presence of ⱖ3 mM zinc, in the presence of acarbose and zinc, one acarbose molecule is
Structure of a (Ca,Zn) Metal Center-containing ␣-Amylase 9879
TABLE I
X-ray data and structure refinement statistics
PWA 䡠 Zn PWA 䡠 Zn (Hg soak) PWA 䡠 Tris PWA 䡠 Ac/Zn
Data collection
Beamlinea X11 BW7A BW7B BW7B
Wavelength (Å)/energy (keV)b 0.9102/13.62 1.0008/12.39 0.8453/14.67 0.8453/14.67
Resolution range (Å) 20.0–2.22 20.0–2.45 99.0–1.52 99.0–1.97
Highest resolution shell (Å) 2.28–2.22 2.51–2.45 1.55–1.52 2.02–1.97
Unit cell dimensions (Å),
space group P212121
a 62.9 62.9 51.5 62.8
b 78.2 78.2 76.5 77.2
c 106.3 106.3 136.5 106.6
No. of unique reflections 26,004 19,410 75,292 35,302
Multiplicity 3.4 3.5 8.5 6.5
具I典/具 (I)典b 10.2 (3.0) 14.7 (4.9) 39.9 (6.1) 18.8 (3.8)
Rsym (%)b,c 6.0 (17.8) 6.5 (21.4) 4.0 (20.9) 7.5 (32.0)
Completenessb 97.7 (93.1) 96.3 (87.4) 89.8 (69.0) 94.4 (88.1)
Refinement
Refinement range (Å) 20–2.2 20–1.6 20–2.0
R factor (%)d 19.0 15.9 19.4
Rfree (%) 22.4 17.2 21.9
No. of non-hydrogen
atoms/asymmetric unit
Total 3975 4252 4007
Protein 3570 3574 3574
bound to the active site at the C-terminal face of the (␣)8- entirely replaced acarbose in the active site of PWA
barrel of domain A. It interacts with a number of highly con- (PWA䡠Tris). Tris is bound by residues that interact with the
served residues that reside in loops connecting -strands 4, 5, valienamine (Arg196, His288, and Asp289) and 4-amino-4,6-
and 7 of the (␣)8-barrel with the subsequent helices (Figs. 2 dideoxy-␣-D-glucose (Glu222 and Asp289) groups of acarbose in
and 3, A and B) (2, 3, 28). Three of its four sugar rings (A–C), the PWA䡠Ac/Zn (Fig. 3, C and D), confirming previous struc-
comprising the acarviosine residue and a linked glucose ring, tural data from a number of ␣-amylase䡠Tris complexes (32,
are visible; but the forth unit accounting for the last glucose 34 –36) and demonstrating its function as a potent competitive
unit at the reducing end of the molecule is not. The location and inhibitor (34, 37, 38).
orientation of the acarbose inhibitor within the active site of In the third crystal form (PWA䡠Zn), zinc binds to the carbox-
PWA resemble previous data from several ␣-amylase䡠acarbose ylate group of the same residue (Glu222) (Fig. 3, E and F; and
complexes. However, in contrast to previous observations (29 – Table II, site 7) that binds to the 4-amino-4,6-dideoxy-␣-D-
33), PWA does not display acarbose transglycosylation activity, glucose group of acarbose (Fig. 3, A and B) and Tris (C and D).
indicating that PWA is not catalytically active under crystalli- The carboxylate group of Asp289 interacts with the zinc ion by
zation conditions. We reasoned that the low temperature used a solvent-mediated hydrogen bond. Comparison of the PWA䡠Zn
for crystal growth of this hyperthermophilic ␣-amylase has and PWA䡠Ac/Zn structures reveals that the position of the zinc
been sufficient to inhibit any transglycolytic catalysis within ion in PWA䡠Zn is nearly identical to that of the acarbose nitro-
the crystal. gen and the partially occupied zinc ion in the PWA䡠Ac/Zn com-
We noticed that residual anomalous difference electron den- plex. However, the PWA䡠Zn structure permits a more detailed
sity remained at the nitrogen position of the 4-amino-4,6- description of the coordination geometry of the zinc ion. Two
dideoxy-␣-D-glucose ring within the active site of the refined solvent molecules are located at positions equivalent to C-7 and
PWA䡠Ac/Zn complex (Fig. 4C). Therefore, a zinc ion was placed O-2 of the valienamine residue in the PWA䡠Ac/Zn complex (Fig.
into this position as an alternative inhibitor (Table II, site 7). 3, A and B). Two additional solvent molecules are located at O-4
Zinc, like the amino group of the 4-amino-4,6-dideoxy-␣-D-glu- and O-6 of the acarbose complex and one near O-3, thus sub-
cose ring of acarbose, is bound by the carboxylate group of stituting the oxygen atoms of the glucose ring of the natural
Glu222. The occupancies of the two inhibitors, acarbose and substrate bound to the ⫺1 subsite. Soaking of the PWA䡠Zn
zinc, were refined to final values of 0.6 and 0.4, respectively. crystal form in a solution containing 15 mM acarbose at pH 6.5
The structural overlay of acarbose and zinc within the active confirmed partial replacement of zinc by the inhibitor (data not
site of the PWA䡠Ac/Zn structure displays directly the competi- shown). Thus, the PWA䡠Zn structure provides, for the first
tive nature of these two chemically unrelated PWA inhibitors time, a molecular rationale for previous observations (10) and
(Figs. 3, A and B; and 4C). On the other hand, if acarbose was our data (Table III) showing how zinc, at concentrations ⬎5
added in 100 mM Tris buffer without zinc, a Tris molecule mM, entirely inhibits the amylolytic activity of PWA. We antic-
9880 Structure of a (Ca,Zn) Metal Center-containing ␣-Amylase
TABLE II observations that magnesium does not inhibit enzyme activity
Metal-binding sites in PWA of different crystal forms (10, 11).
The values for the levels of the metal sites were obtained after
Two Different Metals Bind Near the Active Site—All three
determination of the positive difference peaks (diff) in the Fo ⫺ Fc
difference Fourier electron density maps as described under “Experi- PWA structures contain a two-metal center in close proximity
mental Procedures.” The values for the anomalous peaks (ano) were to the active site cleft, irrespective of specific crystallization
obtained from the anomalous difference electron density maps calcu- conditions and ligand binding to the active site. Therefore, it is
lated using fast Fourier transformation.
most likely that the observed metal ions originate from the
PWA 䡠 Zn PWA 䡠 Ac/Zn PWA 䡠 Tris medium that was used for heterologous expression of PWA in
Site 1 E. coli. The two metal sites are separated by 7.3–7.4 Å (Fig. 4A
Metal Ca Ca Ca and Table II, sites 1 and 2) and are located within the interface
B-factor (Å2) 27.1 24.5 12.4 of domains A and B. Only one of the two sites showed an
(diff/ano) 12.7/– 15.5/– 25.9/–
anomalous signal under the energy conditions used for x-ray
Protein ligands: Asn110, Asp155, Gly157␣, Asp164, Gly202a
Site 2 data collection (Table I), thus indicating a hetero-population of
Metal Zn Zn Zn metals at this site.
B-factor (Å2) 26.8 27.9 12.7 Generally, class-13 glucosidases contain a common calcium-
(diff/ano) 13.0/9.2 24.4/14.1 41.0/59.0
binding site (40), which is essential for their catalytic activity
Protein ligands: His147, His152, Cys166
Site 3 (2, 41). In the PWA structure, this site is matched by a peak
Metal Zn Zn Mg without an anomalous signal, confirming it as common calcium
B-factor (Å2) 27.9 34.9 18.0 site. The calcium ion is coordinated by seven protein ligands in
(diff/ano) 10.2/11.2 16.8/– 11.8/–
a distorted octahedral geometry involving the conserved resi-
Protein ligands: Asp347, Asp349, Glu350
Site 4 due Asn110 from loop 3 (domain A)–1 (domain B), three
residues from the loop preceding ␣-helix 3a of domain A (Asp155
FIG. 3. Active site of PWA with bound acarbose and zinc (PWA䡠Ac/Zn; A and B), Tris (PWA䡠Tris; C and D), and zinc (PWA䡠Zn; E and
F). A, C, and E, structures of active site residues in the presence of bound ligands. Each Fo ⫺ Fc difference electron density map (green) in the
absence of ligands (A, acarbose; C, Tris; and E, zinc) is contoured at 2.0, 2.0, and 4.5 , respectively. In E, the anomalous difference peak (red) is
contoured at 3.7 . B, D, and F, schematic representations of the ligands bound to the active site. Hydrogen bonds are shown by dashed lines. Zinc
ions and solvent molecules are shown as green and gray spheres, respectively. Solvent molecules mediating protein-inhibitor interactions are
indicated in B, D, and F; for clarity, Tyr62, Phe159, and Tyr199 are not shown.
bose-binding site (Ac-IV) is located at the surface of the domain binding at these sites is weaker and less selective than at the
A/C interface (Fig. 1), again with three sugar rings visible. (Ca,Zn) metal center, where the presence/absence of the
These three rings interact with residues from ␣-helix 8b of anomalous difference does not depend on crystallization con-
domain A and -strand 5, the following loop of domain C, and ditions. All three PWA structures display an additional
the C terminus of the protein molecule. metal-binding site (Table II, site 3) at the loop connecting
The presence of an anomalous difference at the remaining -strands 1 and 2 of domain C (Fig. 4B, left panel). In the
metal-binding sites (Table II, sites 3– 6) depends on the pres- PWA䡠Ac/Zn and PWA䡠Zn structures, a zinc ion was modeled
ence of zinc during crystallization, suggesting that metal in this site, which is coordinated by the three carboxylate
9882 Structure of a (Ca,Zn) Metal Center-containing ␣-Amylase
groups of Asp347, Asp349, and Glu350 in a trigonal planar hyperthermostability of PWA and may reflect one strategy of
geometry. In the PWA䡠Ac/Zn structure, this site is also ligan- evolutionary adaptation to a high temperature environment.
ded by an ordered solvent molecule. In the PWA䡠Tris struc-
ture, in contrast, the site bears a magnesium (or calcium) ion DISCUSSION
that is coordinated by the same residue ligands and three Zinc Is a Competitive Active Site Inhibitor That Binds to the
ordered solvent molecules in a distorted octahedral geometry Conserved ⫺1 Active Site Pocket—Acarbose and related sugar
(Fig. 4B, right panel). The structure suggests that this metal- units containing ␣-amylase inhibitors bind to at least three
binding site generally serves a stabilizing role, which may be specific active site pockets in class-13 ␣-amylases. The three
further enhanced by a nearby disulfide bridge connecting PWA structures demonstrate that the ⫺1 pocket of the enzyme
Cys388 and Cys432. Two other metal-binding sites within the is the key site for competitive binding by the unrelated inhib-
interfaces of symmetry-related molecules have been found in itors used in this study. In PWA, this site not only binds
the PWA structures (Fig. 1 and Table II, sites 5 and 6). One organic compounds like acarbose and Tris, but also serves as an
additional metal-binding site in domain A (Table II, site 4) is inhibitory metal-binding site of limited specificity. The two
found only in the presence of zinc, involving at least one chemically related metals Cu2⫹ and Zn2⫹ show comparable
lysine residue as ligand, which is a rare feature in known inhibition of PWA catalysis, suggesting that they indeed bind
protein crystal structures (43). Overall, the total number of into the same pocket and that metal inhibition may correlate
metals coordinated by a single PWA ␣-amylase molecule ex- generally with binding into the ⫺1 active site pocket (Table
ceeds the number of bound metals previously reported for any III). Although the protonation state of the “metal” site in the
other class-13 glycosylhydrolase. The observed high number organic inhibitors (the ternary amino group in Tris and the
of bound metals on the protein surface may enhance the secondary amino group of the 4-amino-4,6-dideoxy-␣-D-glucose
Structure of a (Ca,Zn) Metal Center-containing ␣-Amylase 9883
ring in acarbose) is not directly visible in the PWA structures, Oryza sativa (24% sequence identity) also contains the same
we assume that these groups are protonated and thus are kept molecular arrangement. This sequence motif may also be in-
positively charged by the nearby carboxylate groups of Asp289 dicative of the previously identified close relations between
and Glu222, thereby conferring comparable specific PWA Archaea and plant ␣-amylases (47). The close proximity of the
binding. zinc site of the two-metal center in PWA and the Cys153–Cys154
The ⫺1 pocket residue ligands are highly conserved among disulfide bridge suggests a possible joint requirement for ␣-am-
class-13 ␣-amylase sequences (Fig. 2), indicating that compet- ylase activity under the physiological conditions of P. woesei.
itive metal inhibition could be a general feature of members of However, available cysteine mutations do not influence ther-
this family. To date, no systematic structural and functional mostability and catalytic activity significantly under the con-
studies are available in which the ability of zinc to act as a ditions of the present in vitro measurements (13). Such an
competitive inhibitor of ␣-amylases from different organisms atypical disulfide bridge, connecting residues that are adjacent
was investigated. Interestingly, a molar excess of calcium al- in sequence, is rare in available protein structures. Two of
most completely inhibits the catalytic activity of the ␣-amylase these proteins are members of the alcohol dehydrogenase fam-
from Aspergillus niger (40), whereas it has little effect on the ily, specifically methanol dehydrogenase from Methylobacte-
PWA catalytic activity. These data are reflected by the pres- rium extorquens (48, 49) and ethanol dehydrogenase from
ence of calcium in the ⫺1 active site pocket in the structure of Pseudomonas aeruginosa (50). The atypical disulfide bridge
the ␣-amylase from A. niger (40), whereas no metal ion is found may stabilize the non-planar semiquinone form of the enzyme’s
in the same site in the PWA䡠Tris structure, which was crystal- prosthetic group pyrroloquinoline quinone (49). We speculate
lized in the absence of zinc. Thus, despite the conserved nature that, under the specific living conditions of P. woesei at high
of the ⫺1 active site pocket in ␣-amylases, its affinity for temperatures, rigidification of the active site area by additional
different metal ions may vary. conformational constraints imposed by the presence of such a
The PWA (Ca,Zn)-binding Site Is Essential for PWA Activity, disulfide bridge may be required for in vivo catalytic activity.