From the European Molecular Biology Laboratory,
Notkestrasse 85, D-22603 Hamburg, Germany and the ¶ Department of
Technical Microbiology, Technical University Hamburg-Harburg,
Kasernenstrasse 12, D-21073 Hamburg, Germany
Received for publication, November 6, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The crystal structure of the Besides the recently solved three-dimensional structure of the
glycosyltrehalose trehalosidase from Sulfolobus solfataricus Km1 that exhibits Gene Amplification and Expression--
The wild-type gene
encoding PWA was amplified by PCR using primers deduced from the open
reading frame of the P. furiosus wild-type Purification and Concentration Determination--
The
recombinant Metal Analysis--
The metal content of isolated PWA was
determined by detection of the proton-induced x-ray emission (PIXE) at
the Leipzig 2 MeV proton microprobe (17). Using this technique, all
elements besides those lighter than sodium can be detected in a single scan at a minimum weight limit of ~1-10 ppm. The measurements were
calibrated using the nine sulfur atoms of the PWA sequence (five
cysteines and four methionines) as an internal standard. Protein
samples were dialyzed extensively against Chelex 100-treated buffer
composed of 10 mM sodium phosphate (pH 12) to remove
chloride and sulfur compounds that disturb the protein sulfur signal.
The samples (~2 µl, 1 mg/ml) were dropped onto sample holders
covered with a 0.9 µm polyethylene terephthalate foil. A sample area
of ~1 mm2 was scanned with the proton microbeam, and the
characteristic x-rays were detected using a germanium detector. A total
charge of ~0.17 coulomb was applied within 20 min, ensuring that no
elemental loss (sulfur or metals) due to thermal stress occurred, which would otherwise have changed the x-ray signal intensity. The number of
metals per molecule was calculated from their calibrated signals with
reference to the calibrated sulfur signal. The overall accuracy was
estimated by taking into account the statistical errors of the x-ray
yield and the uncertainty in the correction for x-ray absorption within
the sample.
Crystallization--
Purified recombinant PWA was crystallized
at a concentration of 5.5 mg/ml by the hanging drop vapor diffusion
method. PWA·Zn was grown from 1 µl of protein solution and 2 µl
of 0.1 M MES (pH 6.5) containing 0.01 M
Zn2(SO4)·7H2O and 25% (v/v)
polyethylene glycol monomethyl ether 550 at 19 °C. The
PWA·Ac/Zn complex was grown from 1 µl of acarbose solution (11 mg/ml), 1 µl of protein solution, and 1 µl of 0.1 M
sodium cacodylate (pH 6.5) containing 0.05 M
Zn(OAc)2 and 35% (v/v) 2-methyl-2,4-pentanediol at
25 °C. The PWA·Tris complex was crystallized from 1 µl of
acarbose solution (11 mg/ml), 1 µl of protein solution, and 1 µl of
0.1 M Tris (pH 8.5) containing 0.05 M
MgCl2 and 40% (v/v) ethanol. A heavy atom derivative
suitable for crystallographic phase determination was obtained by
soaking PWA·Zn in a solution containing 29% (v/v) polyethylene
glycol monomethyl ether 550 and 1.0 mM CH3HgCl
for 1.5 h at 19 °C prior to x-ray data collection.
X-ray Data Collection, Processing, and Reduction--
Each x-ray
data collection was performed with one single crystal using 27% (v/v)
polyethylene glycol monomethyl ether 550 as a cryoprotectant for
PWA·Zn. For PWA·Tris, 40% (v/v) ethanol and 31.5% (v/v)
polyethylene glycol 400 were used as a cryoprotectant. For PWA·Ac/Zn,
no transfer into a cryoprotectant was carried out. All crystals were
mounted onto nylon cryo-loops (Hampton Research, Riverside, CA) and
shock-frozen in a nitrogen stream at 100 K. X-ray data sets for the
PWA·Zn, PWA·Ac/Zn, and PWA·Tris complexes were recorded up to
2.2-, 2.0-, and 1.5-Å resolution, respectively. An anomalous
diffraction data set to 2.5-Å resolution was collected from a PWA
mercury derivative isoform at a wavelength above the L-III adsorption
edge of mercury. Further data statistics are presented in Table I. The
data were processed, merged, and scaled using the HKL program suite
(18).
Structure Determination, Refinement, and Model
Evaluation--
Programs used for the subsequent calculations were
from the CCP4 program suite (19), unless stated otherwise. Initial
phases for PWA·Zn were obtained using single isomorphous replacement including the one-wavelength anomalous scattering contribution (SIRAS).
Heavy atom positions were determined from mercury derivative difference
Patterson maps using the RSPS program. From three major binding sites,
initial phases were calculated and refined using SHARP (20) up to
2.5-Å resolution. Phases were improved by solvent flattening with
SHARP. An initial map calculated in CNS (21) was used to build the
model of the molecule using the interactive graphics program O (22).
The model was traced with the help of Bacillus licheniformis
Metal atoms were identified from high peaks in the difference Fourier
maps and in part from additional high peaks in the anomalous difference
Fourier maps. Metals were refined as calcium when no higher peak level
appeared in the anomalous electron density map and the coordination
geometry agreed with that of known protein·calcium complexes. They
were refined as magnesium sites when an excess of magnesium was present
during crystallization and a negative Fo Overall Structure and Ligand Composition--
We have solved the
structure of PWA in three different forms using experimental phases
from a mercury derivative. The structures are in the presence of three
different inhibitors: 1) zinc (PWA·Zn); 2) the specific substrate
analog acarbose and zinc (PWA·Ac/Zn); and 3) Tris, which was used as
a buffer for crystallization trials in the absence of zinc
(PWA·Tris). They have been refined to 2.2-, 2.0-, and 1.6-Å
resolution, respectively. PWA displays the canonical glycosylhydrolase
class-13 fold that is composed of three domains, A-C (Fig.
1). Comparison of the overall structure
of PWA with that of other known
As in other
We initially identified the nature of the bound metal ions by PIXE. For
these experiments, PWA was expressed heterologously in E. coli and purified in the absence of exogenous metals except for
sodium. The PIXE data revealed the presence of 1.1 ± 0.4 molar eq
of zinc and 4 ± 2 molar eq of calcium bound to PWA. In the PWA
crystal structures, the type of the metal sites was characterized by
the analysis of anomalous x-ray data and positive peaks in Fo Chemically Unrelated Inhibitors Competitively Bind to the Active
Site--
We quantified the inhibitory effects of a number of
established
Acarbose is a pseudotetrasaccharide inhibitor consisting of a
valienamine unit at the nonreducing end linked to
4-amino- 4,6-dideoxy-
We noticed that residual anomalous difference electron density remained
at the nitrogen position of the
4-amino-4,6-dideoxy-
In the third crystal form (PWA·Zn), zinc binds to the carboxylate
group of the same residue (Glu222) (Fig. 3, E
and F; and Table II, site 7) that binds to the
4-amino-4,6-dideoxy- Two Different Metals Bind Near the Active Site--
All three PWA
structures contain a two-metal center in close proximity to the active
site cleft, irrespective of specific crystallization conditions and
ligand binding to the active site. Therefore, it is most likely that
the observed metal ions originate from the medium that was used for
heterologous expression of PWA in E. coli. The two metal
sites are separated by 7.3-7.4 Å (Fig. 4A and Table II,
sites 1 and 2) and are located within the interface of domains A and B. Only one of the two sites showed an anomalous signal under the energy
conditions used for x-ray data collection (Table I), thus indicating a
hetero-population of metals at this site.
Generally, class-13 glucosidases contain a common calcium-binding site
(40), which is essential for their catalytic activity (2, 41). In the
PWA structure, this site is matched by a peak without an anomalous
signal, confirming it as common calcium site. The calcium ion is
coordinated by seven protein ligands in a distorted octahedral geometry
involving the conserved residue Asn110 from loop
In contrast to the first metal site (Table II, site 1), there is a
strong anomalous contribution at the second site (site 2). This has
been identified as a zinc site because no other metal with a measurable
anomalous signal under the conditions of x-ray data collection was
found in the PIXE analysis, confirming previous data (13).
The zinc ion is coordinated by ligands in a distorted tetrahedral
geometry, including the imidazole groups of His147 and
His152, the sulfhydryl group of Cys166, and an
ordered solvent molecule. This coordination geometry is typical for
protein·zinc complexes (42, 43). If the cysteine ligand
(Cys166) is replaced, thereby abolishing the zinc site of
this two-metal center, the catalytic activity of PWA at high
temperatures is dramatically reduced, indicating loss of
thermostability (13). Thus, both sites of this two-metal center have a
common role, to stabilize a catalytically active conformation in PWA at
high temperatures.
Additional Ligand-binding Sites--
The high resolution data of
the three PWA structures have allowed the identification of additional
ligand-binding sites (Table II). However, at present, it remains
largely unknown as to whether and to what extent these additional sites
are critical for thermal stability and catalytic function of the
enzyme. Therefore, only a brief account of these sites is given.
Apart from the acarbose site within the PWA active site that is
observed only in the PWA·Ac/Zn structure, three other acarbose sites
have been identified in the two structures in which acarbose was
present in the crystallization medium (PWA·Ac/Zn and PWA·Tris) (Fig. 1). The second acarbose-binding site, with three acarbose rings
visible (Ac-II), is located in a slight depression formed by the
310-helix preceding
The presence of an anomalous difference at the remaining
metal-binding sites (Table II, sites 3-6) depends on the presence of
zinc during crystallization, suggesting that metal binding at these
sites is weaker and less selective than at the (Ca,Zn) metal center,
where the presence/absence of the anomalous difference does not depend
on crystallization conditions. All three PWA structures display an
additional metal-binding site (Table II, site 3) at the loop
connecting Zinc Is a Competitive Active Site Inhibitor That Binds to the
Conserved
The The PWA (Ca,Zn)-binding Site Is Essential for PWA Activity, but Is
Not Conserved--
We have solved the first structure of an
To date, only two other structures of
Another specific feature of the PWA structure is the presence of a
disulfide bond in domain B that is formed by two adjacent cysteines,
Cys153 and Cys154. This sequence motif is
confined to a limited number of Archaea Implications for Biotechnological Processes--
Amylases,
along with other starch-hydrolyzing enzymes like pullulanases and
glucoamylases, have widespread applications in the food, chemical, and
pharmaceutical industries (4, 6) and compose ~30% of the current
industrial enzyme production. Some of these processes, such as the
liquefaction of starch, require high temperatures of up to 100 °C.
At present, mostly the
Not only is PWA superior to other -amylase from the
hyperthermophilic archaeon Pyrococcus woesei was
solved in the presence of three inhibitors: acarbose, Tris, and zinc.
In the absence of exogenous metals, this
-amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a
novel, activating, two-metal (Ca,Zn)-binding site and a second
inhibitory zinc-binding site that is found in the
1 sugar-binding
pocket within the active site. The data resolve the apparent paradox
between the zinc requirement for catalytic activity and its strong
inhibitory effect when added in molar excess. They provide a rationale
as to why this
-amylase, in contrast to commercially available
-amylases, does not require the addition of metal ions for full
catalytic activity, suggesting it as an ideal target to maximize the
efficiency of industrial processes like liquefaction of starch.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amylases (
-1,4-D-glucan 4-glucanohydrolase, EC
3.2.1.1) are endo-acting hydrolases that randomly cleave the
-1,4-glycosidic linkages of branched and linear carbohydrates such
as amylopectin and amylose in starch and glycogen (1-3). Their
widespread occurrence in various organisms and the consumption of their
substrates for food reserves and energy sources have led to intense
interest in their biomedical properties and to major biotechnological
applications in industry (4-6). Their value in specific industrial
processes depends critically on their pH and optimal temperature, which vary depending on the organism of origin. Nearly all
-amylases belong to class-13 of glycosylhydrolases by virtue of their
characteristic sequence motifs. However, in general, their overall
sequence similarity is low, which, for some members, falls below the
10% level (1-3, 7). A number of crystal structures of class-13
members have been solved, comprising
-amylases from organisms
inhabiting environments that span a large temperature range, from
psychrophilic to hyperthermophilic (3). These
-amylases share a
common three-domain fold with the catalytic activity located on the
C-terminal face of a central (
)8-barrel, domain A. The sequences of the other two domains, B and C, are void of any
conserved motifs and are not involved directly in substrate catalysis.
-amylase activity (8), no other structures of
archael class-13
-amylases are known to date. The
-amylase from
the hyperthermophilic archaeon Pyrococcus woesei
(PWA),1 which is identical to
that from Pyrococcus furiosus (9), was cloned and classified
as a class-13 glycosylhydrolase (10). Throughout this study, both the
-amylases of P. woesei and P. furiosus
will be referred to as PWA. Biochemical characterization of recombinant
PWA revealed a high thermal stability and maximum catalytic activity at
~100 °C (10-12). In a recent study using coupled plasma-atomic
emission spectroscopy, it was shown that PWA binds calcium and zinc
stoichiometrically and with high affinity (13). Thus, in contrast to
many other
-amylases, PWA activity does not require the addition of
metal ions. Due to its superior properties, exceeding those of
-amylases currently employed in commercial preparations (5, 14), PWA
has become a prime candidate for maximizing the efficiency of
applications in the starch industry. To reveal the molecular basis of
its properties, we solved the x-ray structure of PWA in the presence of
three different active site ligands. The structure reveals a novel
(Ca,Zn)-binding site in close proximity to the active site cleft, which
is not found in
-amylases of any bacteria, plants, and most other
Archaea. The presence of this site indicates adaptive evolution of PWA to the specific living conditions of P. woesei. The three
complex structures also show how competitive binding of organic
compounds and zinc provides a direct molecular explanation as to why a
molar excess of zinc or some chemically related metals inhibits PWA activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase gene
(10). An NcoI recognition site (underlined) was fused to the
5'-end of the sense primer (5'-CAT GCC ATG GAC ATA AAG AAA TTA ACA CCC CTC-3'), as was an XhoI recognition
site (underlined) to the antisense primer (5'-GGC CTC GAG
TCA CCC AAC ACC ACA ATA ACT CCA-3'). Additionally, the natural GTG
start codon was replaced with the Escherichia coli start
codon ATG (boldface). The PCR product was digested twice with
NcoI/XhoI and cloned into the
NcoI/XhoI cloning site of the pET-15b vector
(Novagen, Madison, WI) to generate the plasmid pPWA. pPWA was cloned
and expressed in E. coli strain BL21(DE3) (15) as described
previously (12). The deduced amino acid sequence is identical to the
-amylase gene from P. furiosus (10) and to the P. woesei amylase sequence released to the NCBI Protein Database
by Lu et al.2
-amylase gene product was purified from catalytically
active inclusion bodies (12). Inclusion bodies were solubilized in 0.12 M Britton and Robinson buffer (pH 12) and purified
by hydrophobic interaction chromatography using a phenyl-Superose HR
5/5 column (Amersham Biosciences, Freiburg, Germany). The protein was
eluted in 0.02 M triethanolamine containing 30% (v/v)
isopropyl alcohol (pH 7.2). Fractions containing amylolytic activity at 99 °C were pooled and further purified by size-exclusion
chromatography using a Superdex 200 16/60 prep-grade column. The
amylase was eluted in 0.02 M triethanolamine (pH 7.2) and
concentrated to a final concentration of 5.5 mg/ml in an Amicon
ultrafiltration device (Millipore Corp., Eschborn, Germany). The
concentration of the enzyme preparation was determined by estimating
the extinction coefficient according to the method of Gill and von
Hippel (16).
-amylase (BLA) as a template. The final model was built in a
consecutive cycle of crystallographic refinement using CNS and manual
rebuilding. A total number of 25,967 reflections in the resolution
range of 20-2.22 Å were included in the refinement, with 3.8% (995 reflections) set aside for cross-validation (23). The initial
R-value was 44.2%, and the free R-factor was
42.1%. After several cycles of positional and restrained individual
B-factor refinement, solvent building was performed using
the solvent 0 mode of ArpWArp (24) and CNS with waters placed in local
maxima of difference electron density maps above 3
. The structures of the two PWA·inhibitor complexes (PWA·Tris and PWA·Ac/Zn) were solved by molecular replacement using the structure of PWA·Zn as a
search model. Rotation and translation functions were calculated with
AMoRe (25) using x-ray data from 10 to 2.5 Å, resulting in a single
solution with correlation coefficients of 68.6 and 80.1% and
R-factors of 39.4 and 33.5% for PWA·Tris and PWA·Ac/Zn, respectively. A test set of 1686 reflections (4.7%, PWA·Tris) and
1196 reflections (1.7%, PWA·Ac/Zn) for the calculation of Rfree was excluded during refinement in CNS.
Initial phases of the PWA·Tris model were applied to automated
model building using ArpWArp for model completion. The models were
verified and corrected, and acarbose and Tris molecules were built
manually using the graphics software program O. Solvent building and
subsequent refinement were performed as described above.
Fc difference Fourier electron density peak appeared
when the metal position was refined as calcium. Metals were refined as
zinc when the crystal growth conditions contained zinc ions and if the
metals sites showed a high anomalous peak emerging from data set
collection at 12.4 keV (
= 0.91 Å), which is above the
k-adsorption edge of zinc of 9.59 keV (
= 1.28 Å).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylases using the program DALI
(26) revealed the highest structural similarity to the homologous
enzymes from the hyperthermophile B. licheniformis (BLA;
29% sequence identity; root mean square deviation of 2.5 Å based on
comparison of the C
backbone trace) and from barley
(28% sequence identity; root mean square deviation of 1.8 Å) (Fig.
2).
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Fig. 1.
Overall crystal structure of PWA complexed
with different ligands. The cylinders and
arrows represent helices and -strands, respectively.
Domains A-C are colored cyan, magenta, and
brown, respectively. For clarity, the
-strands composing
the
-barrel of domain A are colored gray. Bound metals
(zinc, green; and magnesium and calcium, orange)
and acarbose molecules are indicated and numbered according to Table
II. The backbone of the acarbose molecules is yellow; oxygen
atoms are red; and nitrogen atoms are blue.
A, top view of the PWA·Ac/Zn complex. The active site
cleft at the front face of the PWA molecule contains two inhibitors
with partial occupancies, the first of which is acarbose and the second
of which is a coordinated zinc ion, which is virtually identical
to the nitrogen position of the
4-amino-4,6-dideoxy-
-D-glucose ring of acarbose. The two
inhibitors are superimposed onto each other. B, PWA·Ac/Zn
rotated 90° along a horizontal axis with respect to the orientation
in A. C, PWA·Tris shown from the same
orientation as in A. Three zinc-binding sites
(Zn3, Zn5, and Zn6) of PWA·Ac/Zn are
replaced by magnesium ions in PWA·Tris (Mg3,
Mg5, and Mg6). In PWA·Tris, no metal is found
in site 4 of the PWA·Ac/Zn structure.
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Fig. 2.
Structure-based alignment of PWA
sequences. The secondary structure assignments for PWA are shown
as colored cylinders (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.
-amylases, the central domain A, covering residues
1-109 and 170-340, is folded as a (
)8-barrel and
contains the active site at its C-terminal face. Domain B (residues
111-169) inserts between
-strand 3 and
-helix 3a of domain A,
thus forming part of the active site cleft. Its secondary structure is
limited to two short
-strands, forming a small antiparallel
-sheet and a short 310-helix (Figs. 1 and 2). At their
interface, domains A and B comprise a novel (Ca,Zn) metal center, which
is described further below (see Fig. 4). A disulfide bridge is formed
by two consecutive cysteine residues (Cys153 and
Cys154) in close vicinity to the zinc-binding site of this
two-metal center. Covering a range of 58 residues only, domain B of PWA is one of the smallest
-amylase B domains, whereas other members of
the family span >100 residues (2, 3). Among those with a known
structure, domain B of BLA is most similar (root mean square deviation
of 2.3 Å based on the C
backbone) (27) to the
corresponding domain of PWA. In contrast, domain B of BLA contains 43 additional residues that form a second
-sheet (27). The C-terminal
domain C (residues 341-435) is arranged in an eight-stranded
antiparallel
-sheet containing a Greek key motif. The
function of this domain still remains unclear.
Fc difference Fourier
electron density maps (Table I). Based on
these data, the PWA structures show excessive metal binding; all three
structures have five metal-binding sites in common (Table
II). The two crystal forms grown in the presence of zinc (PWA·Zn and PWA·Ac/Zn) reveal two additional metal
sites, bringing the total in these structures to seven. The presence of
an anomalous signal under the experimental conditions of x-ray data
collection was used to indicate the presence of zinc (Tables I and II).
In the PWA·Tris structure, solved from crystals grown in the absence
of zinc, but in the presence of magnesium, only one site showed a
significant anomalous signal and therefore was refined as a zinc site
(Table II). The remaining four sites, without an anomalous signal, but
retaining strong positive difference electron density, when refined as
an ordered solvent (>10
), were interpreted to be calcium or
magnesium sites. Thus, the stoichiometry of calcium (or magnesium) and
zinc sites observed in the PWA·Tris structure is in good agreement
with the PIXE data. In contrast, in the PWA·Zn and PWA·Ac/Zn
structures, all sites except one showed a significant anomalous signal
and were therefore refined as zinc sites. The remaining site was
interpreted as a calcium site. These metal sites will be referred to as
activating sites (Table II, sites 1 and 2), an inhibiting site (site
7), and other sites (sites 3-6).
X-ray data and structure refinement statistics
Metal-binding sites in PWA of different crystal forms
levels of the metal sites were obtained after
determination of the positive difference peaks (diff) in the
Fo
Fc difference Fourier
electron density maps as described under "Experimental Procedures."
The
values for the anomalous peaks (ano) were obtained from the
anomalous difference electron density maps calculated using fast
Fourier transformation.
-amylase active site ligands, including the transition
state analog acarbose and the buffer Tris (Table
III). To link the observed tight binding
of calcium and zinc to a potential role in activity regulation, we also
assessed the regulatory properties of a number of metal ions (Table
III). Except for copper (full inhibition in the presence of 3 mM Cu2+), zinc had the strongest inhibitory
effect. In the presence of
3 mM zinc, residual PWA
activity was <10%. In contrast to many other characterized
-amylases, including commercially available BLA (10, 13), PWA was
not activated significantly by an excess of calcium (Table III). Based
on the measured inhibition data, we selected three ligands (acarbose,
Tris, and zinc) for crystallographic characterization of the PWA active
site (Fig. 3).
Effect of metal ions and chemical reagents on PWA activity
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Fig. 3.
Active site of PWA with bound acarbose and
zinc (PWA·Ac/Zn; A and B), Tris
(PWA·Tris; C and D), and zinc
(PWA·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.
-D-glucose, which is fused to
maltose. In the PWA·Ac/Zn structure, resulting from
co-crystallization of PWA in the presence of acarbose and zinc, one
acarbose molecule is bound to the active site at the C-terminal face of
the (
)8-barrel of domain A. It interacts with a
number of highly conserved residues that reside in loops connecting
-strands 4, 5, and 7 of the (
)8-barrel with the subsequent helices (Figs. 2 and 3, A and B) (2,
3, 28). Three of its four sugar rings (A-C), comprising the
acarviosine residue and a linked glucose ring, are visible; but
the forth unit accounting for the last glucose unit at the reducing end of the molecule is not. The location and orientation of the acarbose inhibitor within the active site of PWA resemble previous data from
several
-amylase·acarbose complexes. However, in contrast to
previous observations (29-33), PWA does not display acarbose transglycosylation activity, indicating that PWA is not catalytically active under crystallization conditions. We reasoned that the low
temperature used for crystal growth of this hyperthermophilic
-amylase has been sufficient to inhibit any transglycolytic
catalysis within the crystal.
-D-glucose ring within the active
site of the refined PWA·Ac/Zn complex (Fig. 4C). Therefore, a zinc ion was
placed into this position as an alternative inhibitor (Table II, site
7). Zinc, like the amino group of the
4-amino-4,6-dideoxy-
-D-glucose ring of acarbose, is
bound by the carboxylate group of Glu222. The occupancies
of the two inhibitors, acarbose and zinc, were refined to final values
of 0.6 and 0.4, respectively. The structural overlay of acarbose and
zinc within the active site of the PWA·Ac/Zn structure displays
directly the competitive nature of these two chemically unrelated PWA
inhibitors (Figs. 3, A and B; and 4C). On the other hand, if acarbose was added in 100 mM Tris
buffer without zinc, a Tris molecule entirely replaced acarbose in the active site of PWA (PWA·Tris). Tris is bound by residues that interact with the valienamine (Arg196, His288,
and Asp289) and
4-amino-4,6-dideoxy-
-D-glucose (Glu222 and
Asp289) groups of acarbose in the PWA·Ac/Zn (Fig. 3,
C and D), confirming previous structural data
from a number of
-amylase·Tris complexes (32, 34-36) and
demonstrating its function as a potent competitive inhibitor (34, 37,
38).
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Fig. 4.
Unique metal-binding sites in PWA.
A, structure of the two-metal (Ca,Zn)-binding site in PWA
(cf. Table II, sites 1 and 2). The zinc ion (gray
sphere) is coordinated by two histidines (His147 and
His152), one cysteine (Cys166), and an ordered
solvent molecule (red). The calcium ion is coordinated by
the conserved residues Asn110, Asp155,
Gly157, Asp164, and Gly202
(cf. Fig. 2) and a solvent molecule. The
Fo Fc difference and anomalous
difference maps are colored as described in the legend to Fig. 3 and
contoured at 4.5 and 33
, respectively. B, structure of
the variable PWA metal-binding site in domain C (cf. Table
II, site 3). Left panel, zinc (green sphere) is
coordinated by three negatively charged amino acid side chains
(Asp347, Asp349, and Glu350) and a
solvent molecule (red sphere). Right panel, in
the PWA·Tris structure, a magnesium ion is coordinated by the same
side chain residues as zinc in the left panel. However, two
additional solvent molecules lead to a different overall coordination
geometry. C, shown is the structure of the partially
occupied inhibitory zinc-binding site in the
1 active site
pocket of PWA, which is superimposed on the bound acarbose ligand. The
residual anomalous difference electron density at the zinc position is
shown at a contour level of 3.2
.
-D-glucose group of acarbose (Fig.
3, A and B) and Tris (C and
D). The carboxylate group of Asp289 interacts
with the zinc ion by a solvent-mediated hydrogen bond. Comparison of
the PWA·Zn and PWA·Ac/Zn structures reveals that the position of
the zinc ion in PWA·Zn is nearly identical to that of the acarbose
nitrogen and the partially occupied zinc ion in the PWA·Ac/Zn
complex. However, the PWA·Zn structure permits a more detailed
description of the coordination geometry of the zinc ion. Two solvent
molecules are located at positions equivalent to C-7 and O-2 of the
valienamine residue in the PWA·Ac/Zn complex (Fig. 3, A
and B). Two additional solvent molecules are located at O-4
and O-6 of the acarbose complex and one near O-3, thus substituting the
oxygen atoms of the glucose ring of the natural substrate bound to the
1 subsite. Soaking of the PWA·Zn crystal form in a solution
containing 15 mM acarbose at pH 6.5 confirmed partial
replacement of zinc by the inhibitor (data not shown). Thus, the
PWA·Zn structure provides, for the first time, a molecular rationale
for previous observations (10) and our data (Table III) showing how
zinc, at concentrations >5 mM, entirely inhibits the
amylolytic activity of PWA. We anticipate that the strong inhibition of
many
-amylases by zinc (39) is via the same binding to the
1 site
as observed in PWA·Zn. In contrast, the PWA structures do not
indicate that magnesium binds to the active site, confirming previous
biochemical observations that magnesium does not inhibit enzyme
activity (10, 11).
3
(domain A)-
1 (domain B), three residues from the loop preceding
-helix 3a of domain A (Asp155 as bidendate ligand,
Gly157, and Asp164), Gly202 from a
310-helix between
-strand 4 and
-helix 4 of
domain A, and one ordered solvent molecule (Fig. 4A). Thus,
the number of protein ligands in this calcium-binding site exceeds
those found for the same site in other class-13
-amylases (2).
-strand 1 of domain A, the loop
connecting
-helix 6b and
-strand 7 of domain A, and the loop
connecting
-helix 8b of domain A and the first
-strand of domain
C (Fig. 1). A similar carbohydrate-binding site was reported for a
chimeric Bacillus amyloliquefaciens/licheniformis
-amylase (32). The third acarbose-binding site (Ac-III) is located
at the surface of domain B at a distance of ~30 Å from the active
site (Fig. 1). This site corresponds to the so-called accessory
carbohydrate site reported for the pig pancreas
-amylase structure
(44). The fourth acarbose-binding site (Ac-IV) is located at the
surface of the domain A/C interface (Fig. 1), again with three sugar
rings visible. These three rings interact with residues from
-helix 8b of domain A and
-strand 5, the following loop of domain C, and
the C terminus of the protein molecule.
-strands 1 and 2 of domain C (Fig. 4B,
left panel). In the PWA·Ac/Zn and PWA·Zn structures, a
zinc ion was modeled in this site, which is coordinated by the three
carboxylate groups of Asp347, Asp349, and
Glu350 in a trigonal planar geometry. In the PWA·Ac/Zn
structure, this site is also liganded by an ordered solvent molecule.
In the PWA·Tris structure, in contrast, the site bears a magnesium
(or calcium) ion that is coordinated by the same residue ligands and
three ordered solvent molecules in a distorted octahedral geometry
(Fig. 4B, right panel). The structure suggests
that this metal-binding site generally serves a stabilizing role, which
may be further enhanced by a nearby disulfide bridge connecting
Cys388 and Cys432. Two other metal-binding
sites within the interfaces of symmetry-related molecules have been
found in the PWA structures (Fig. 1 and Table II, sites 5 and 6). One
additional metal-binding site in domain A (Table II, site 4) is found
only in the presence of zinc, involving at least one lysine residue as
ligand, which is a rare feature in known protein crystal structures
(43). Overall, the total number of metals coordinated by a single PWA
-amylase molecule exceeds the number of bound metals previously
reported for any other class-13 glycosylhydrolase. The observed high
number of bound metals on the protein surface may enhance the
hyperthermostability of PWA and may reflect one strategy of
evolutionary adaptation to a high temperature environment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 Active Site Pocket--
Acarbose and related sugar units
containing
-amylase inhibitors bind to at least three specific
active site pockets in class-13
-amylases. The three PWA structures
demonstrate that the
1 pocket of the enzyme is the key site for
competitive binding by the unrelated inhibitors used in this study. In
PWA, this site not only binds organic compounds like acarbose and Tris,
but also serves as an inhibitory metal-binding site of limited
specificity. The two chemically related metals Cu2+ and
Zn2+ show comparable inhibition of PWA catalysis,
suggesting that they indeed bind into the same pocket and that metal
inhibition may correlate generally with binding into the
1 active
site pocket (Table III). Although the protonation state of the
"metal" site in the organic inhibitors (the ternary amino group in
Tris and the secondary amino group of the
4-amino-4,6-dideoxy-
-D-glucose ring in acarbose) is not
directly visible in the PWA structures, we assume that these groups are
protonated and thus are kept positively charged by the nearby
carboxylate groups of Asp289 and Glu222,
thereby conferring comparable specific PWA binding.
1 pocket residue ligands are highly conserved among class-13
-amylase sequences (Fig. 2), indicating that competitive metal
inhibition could be a general feature of members of this family. To
date, no systematic structural and functional studies are available in
which the ability of zinc to act as a competitive inhibitor of
-amylases from different organisms was investigated. Interestingly,
a molar excess of calcium almost completely inhibits the catalytic
activity of the
-amylase from Aspergillus niger (40),
whereas it has little effect on the PWA catalytic activity. These data
are reflected by the presence of calcium in the
1 active site pocket
in the structure of the
-amylase from A. niger (40),
whereas no metal ion is found in the same site in the PWA·Tris
structure, which was crystallized in the absence of zinc. Thus, despite
the conserved nature of the
1 active site pocket in
-amylases, its
affinity for different metal ions may vary.
-amylase that comprises a mixed (Ca,Zn) two-metal center in close
proximity to the active site cleft. The three structures in the
presence of different inhibitors (zinc, acarbose/zinc, and Tris)
provide a molecular rationale for previous biochemical analyses of PWA
(13) and our current data (Table III) indicating specific and tight
binding of zinc and calcium. If the zinc site of this two-metal center is abolished by replacing its cysteine ligand (Cys166), the
catalytic activity of PWA at high temperatures is dramatically reduced,
indicating loss of thermostability (13). Comparison of
-amylase
sequences (data not shown) and use of Cys166 as an
indicator denote that the zinc site of the two-metal center is present
only in P. woesei and its close homolog Thermococcus hydrothermalis (84% sequence identity). Even in the more closely related Archaea sequences from Pyrococcus kodakaraensis and
Thermococcus sp., this cysteine is replaced by an alanine,
indicating that this site is lost.
-amylases with a two-metal
center are available. One belongs to the hyperthermophile B. licheniformis, which binds two calcium ions that are probably bridged by a sodium ion (27). This site superimposes well with the
(Ca,Zn) two-metal center of PWA (Fig. 4A). Both two-metal centers share the conserved calcium-binding site (site I in BLA) (27),
which is common to many class-13
-amylases, whereas the coordination
geometry of the second differs. These data indicate divergent
evolutionary paths for the adaptation of
-amylases in Archaea
(P. woesei) and bacteria (B. licheniformis;
Topt = 90 °C) to high temperature
environments. As such, these
-amylases have evolved as either a
homo-(Ca,Ca)- or a hetero-(Ca,Zn) two-metal center, respectively.
The other known
-amylase structure with a two-metal center is
the meso-stable
-amylase from barley, which shares the
highly conserved calcium-binding site and displays an additional
calcium-binding site at a distance of ~7 Å (45, 46). However, in
contrast to the two calcium sites in BLA, the second calcium site of
the barley
-amylase structure does not superimpose with the second
site of the (Ca,Zn) two-metal center in PWA.
-amylase sequences,
including those of P. kodakaraensis, T. hydrothermalis, and Thermococcus sp. One loosely
related plant
-amylase sequence from Oryza sativa (24%
sequence identity) also contains the same molecular arrangement. This
sequence motif may also be indicative of the previously identified
close relations between Archaea and plant
-amylases (47). The close
proximity of the zinc site of the two-metal center in PWA and the
Cys153-Cys154 disulfide bridge suggests a
possible joint requirement for
-amylase activity under the
physiological conditions of P. woesei. However, available
cysteine mutations do not influence thermostability and catalytic
activity significantly under the conditions of the present in
vitro measurements (13). Such an atypical disulfide bridge,
connecting residues that are adjacent in sequence, is rare in available
protein structures. Two of these proteins are members of the alcohol
dehydrogenase family, specifically methanol dehydrogenase from
Methylobacterium extorquens (48, 49) and ethanol
dehydrogenase from Pseudomonas aeruginosa (50). The atypical
disulfide bridge may stabilize the non-planar semiquinone form of the
enzyme's prosthetic group pyrroloquinoline quinone (49). We speculate
that, under the specific living conditions of P. woesei at
high temperatures, rigidification of the active site area by additional
conformational constraints imposed by the presence of such a disulfide
bridge may be required for in vivo catalytic activity.
However, the precise molecular role of this disulfide bridge in PWA
substrate catalysis with respect to thermostability still remains to be
defined experimentally.
-amylases from B. licheniformis
and B. stearothermophilus are used commercially in the
liquefaction process of starch due to their high thermostability (5).
However, the
-amylases from these organisms display full catalytic
activity and stability only if calcium is added. Unfortunately, the
addition of calcium inhibits glucoamylases and destabilizes glucose
isomerases, which are used in subsequent starch-processing reactions,
thus stimulating investigations into alternative
-amylases that do
not require addition of metal ions during enzymatic processes at the
industrial scale.
-amylases with respect to
thermostability, but it also lacks an exogenous calcium requirement for
full catalytic activity (Table III) (10, 13). Therefore, PWA has been
proposed as an alternative to BLA to further improve the efficiency of
industrial processes in starch liquefaction (5, 6). In this work, we
have solved the PWA crystal structures and revealed the molecular basis
for tight calcium and zinc binding by the identification of a
nonconserved and PWA-specific (Ca,Zn) two-metal center. Although in all
structures of glycosylhydrolase class-13 amylases known so far, the
calcium in domain B is coordinated by not more than six protein
ligands, in PWA, it is coordinated by seven protein ligands, thus
providing a structural rationale for the high binding affinity of the
latter enzyme. In addition, the PWA structure reveals a novel
zinc-binding site in close proximity to the calcium-binding site
previously established to be essential for catalytic activity and
stability (13). Activation of PWA by traces of zinc is, however,
superseded by the competitive active site inhibitory effects of this
metal. P. woesei has evolved this structural property as a
unique evolutionary adaptation most probably to retain full PWA
activity in its extremophilic living condition, utilizing zinc as a
positive and negative regulator in a
concentration-dependent manner. The PWA structures offer a
strong base upon which to further engineer properties of PWA that are
more conducive to potential applications in industrial processes. In
particular, they reveal two metal-binding sites (zinc and calcium) with
different functions, which are well suited to optimize the properties
of PWA for biotechnological applications.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank E. Möller (Bayer AG, Leverkusen, Germany) for kindly providing acarbose and D. S. Auld for helpful discussions on the interpretation of metal-binding sites.
![]() |
FOOTNOTES |
---|
* 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 atomic coordinates and the structure factors (code 1MWO, 1MXD, and 1MXG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
To whom correspondence should be addressed: EMBL, Hamburg
Outstation, c/o DESY, Notkestr. 85, D-22603 Hamburg, Germany. Tel.: 49-40-89902-126; Fax: 49-40-89902-149; E-mail:
wilmanns@embl-hamburg.de.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211339200
2 C. Lu, J. Weizheng, and Y. Yunyan, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PWA, P.
woesei -amylase;
PIXE, proton-induced x-ray emission;
MES, 4-morpholineethanesulfonic acid;
Ac, acarbose;
BLA, B.
licheniformis
-amylase.
![]() |
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