From the Protein Structure Group, Department of
Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100
Copenhagen, Denmark and the ¶ Département de Génie
Biochimique et Alimentaire, Centre de BioIngéniere G. Durand,
UMR CNRS 5504, UMR INRA 792, Institut National des Sciences Appliquees,
Avenue de Rangueil, F-31077 Toulouse Cedex 4, France
Received for publication, December 6, 2000, and in revised form, March 30, 2001
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ABSTRACT |
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Amylosucrase (E.C. 2.4.1.4) is a member of Family
13 of the glycoside hydrolases (the Amylosucrase (AS)1 is a
hexosyltransferase (E.C. 2.4.1.4) produced by non-pathogenic
bacteria from the Neisseria genus and was identified in
N. perflava as early as 1946 (1). MacKenzie et
al. (2) identified intracellular AS in six other
Neisseriae species, and later an extracellular N. polysaccharea AS was discovered (3). N. polysaccharea
was isolated from the throats of healthy children, and it was suggested
that the function of the secreted glucansucrase AS was to produce
insoluble polymers. Until recently AS has only been found in bacteria
from the Neisseria genus, but the Deinococcus
radiodurans genome (4) and the Caulobacter crescentus
genome (5) actually encodes proteins with a similar length that are 43 and 34%, respectively, identical to AS from N. polysaccharea.
In the presence of an activator polymer (e.g. glycogen), AS
catalyzes the synthesis of an amylose-like polysaccharide composed of
only The recent cloning of N. polysaccharea AS in E. coli (8, 9) has made mutational (10) and detailed kinetic studies of highly purified enzyme possible (11, 12). It has also provided AS in
sufficient amounts for successful crystallization experiments (13). The
recombinant AS is derived from a glutathione S-transferase fusion protein and consists of a single polypeptide chain with 636 amino acid residues including 6 cysteines and 15 methionines.
Based on amino acid sequence comparisons, AS has been suggested to
belong to the The -amylases), although its
biological function is the synthesis of amylose-like polymers from
sucrose. The structure of amylosucrase from Neisseria
polysaccharea is divided into five domains: an all helical
N-terminal domain that is not similar to any known fold, a
(
/
)8-barrel A-domain, B- and B'-domains displaying
/
-structure, and a C-terminal eight-stranded
-sheet domain. In
contrast to other Family 13 hydrolases that have the active site in the
bottom of a large cleft, the active site of amylosucrase is at the
bottom of a pocket at the molecular surface. A substrate binding site
resembling the amylase 2 subsite is not found in amylosucrase. The site
is blocked by a salt bridge between residues in the second and eight
loops of the (
/
)8-barrel. The result is an exo-acting
enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with
the loop structure found in other hydrolases, and this insertion
(forming domain B') is suggested to be important for the polymer
synthase activity of the enzyme. The topology of the B'-domain
creates an active site entrance with several ravines in the molecular
surface that could be used specifically by the substrates/products
(sucrose, glucan polymer, and fructose) that have to get in and out of
the active site pocket.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-(1
4)-glucosidic linkages using sucrose as the only energy
source (6). This glycogen pathway is not found in e.g. Escherichia coli, which like most bacteria require activated
-D-glucosyl-nucleoside-diphosphate substrates for
polysaccharide synthesis (7). The utilization of a readily available
substrate makes AS a potentially very useful glucosylation tool for the
production of novel amylopolysaccharides.
-amylase superfamily, the
-retaining glycoside hydrolase (GH) Family 13 (14). Putative active site residues in the
predicted (
/
)8-barrel have also been pointed out (9). Most members of this family hydrolyze
-(1
4) and
-(1
6)-glucosidic linkages of starch.
-amylase reaction mechanism is a general acid catalysis, similar
to all of the glucoside hydrolases (16), and the same mechanistic
scheme can also accommodate glucan synthesis from sucrose as shown in
Scheme 1. The reaction is initiated by
simultaneous protonation of the glycosidic bond by a proton donor and a
nucleophilic attack on the anomeric carbon of the glucose moiety. This
leads to the covalently linked substrate-enzyme intermediate. The
intermediate can react with either water or with another saccharide
molecule, as shown in the scheme. This implicates that the ratio
between hydrolysis and transglycosylation is determined only by the
relative concentrations of water and sugar moieties in the active
site.
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Scheme 1.
General reaction mechanism for hydrolysis
and transglycosylation.
Consistent with Scheme 1, AS catalyzes both sucrose hydrolysis and oligosaccharide and polymer synthesis in the absence of an activator polymer (11). With 10 mM sucrose as the sole substrate, AS produces glucose (30%), maltose (29%), maltotriose (18%), turanose (11%), and insoluble polymer (12%).
Apart from AS, the GH Family 13 comprises other enzymes with
non-hydrolytic functions. The crystal structure of a cyclodextrin glucanotransferase (17) (CGTase) showed an active site architecture very similar to the -amylase from Aspergillus oryzae (18)
(TAKA-amylase, the first
-amylase structure determined) and thus
implicated very similar reaction mechanisms at least for the formation
of the covalent intermediate. The presence of a covalent intermediate has been verified experimentally in CGTases (19).
These findings all suggest that the active site of AS is highly similar
to those of the -amylases and CGTases. Structure determinations of
complexes with substrate analogues have yielded detailed information on
the
-amylase structure/function relationships. A stringent
nomenclature for enzyme-substrate interactions has been developed, and
substrate binding is usually described in terms of numbered sugar
binding subsites (20). The catalytic residues are then located between
the sugar binding subsites
1 and +1, when numbering the
polysaccharide from the reducing end. In this work, we present the
crystal structure of AS at a resolution of 1.4 Å, which represents the
first crystal structure of a glucansucrase and is the first structure
of a glucan-elongating enzyme from the GH 13 family. The structural
alignment of AS and
-amylase-substrate analogue complexes is well
suited to provide a basis for the understanding of product profile and
substrate specificity observed for AS.
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EXPERIMENTAL PROCEDURES |
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Crystallization--
Expression and purification of recombinant
AS was performed as described previously (9, 12). The production of
Se-Met AS and the crystallization conditions (equal amounts of 4 mg/ml protein solution (150 mM NaCl, 50 mM Tris-HCl,
pH 7.0, 1 mM EDTA, and 1 mM -dithiothreitol)
and reservoir solution (30% polyethylene glycol
Mr 6000 and 0.1 M HEPES, pH 7.0))
have been published (13).
Data Collection, Structure Determination, and
Refinement--
All data were collected at the ESRF, Grenoble and were
processed and scaled using DENZO and SCALEPACK (21). Multiple
wavelength anomalous dispersion (MAD) data were collected at beamline
BM 14. Data were collected using energies corresponding to the
inflection point and peak of the experimentally determined selenium K
edge and a remote high energy wavelength (Table
I). All 15 selenium sites were identified
when the MAD data were analyzed by the SOLVE program (22). Phases were
extended to 1.7 Å using DM (23), and the structure was build with the
automated tracing procedure ARP/wARP (24). This tracing located 625 of
the total 628 amino acid residues found in the structure. Later a 1.4 Å native data set was obtained at beamline ID 14 EH 1 ( = 0.934 Å), and the structure was refined at this level of resolution.
Further rebuilding was done in program O (25), and refinement was
performed with the CNS program package (26). A total of 628 amino acid
residues, one Tris molecule, one HEPES molecule, a sodium ion, and 751 water molecules were included in the final model. Refinement statistics are listed in Table I. Several patches of elongated electron density
that could arise from a polyethylene glycol molecule were not fitted.
Thirty-one of the side chains were fitted with two conformations, and
for sixteen surface side chains some of the outermost atoms displayed
high B-factors. A polymerase chain reaction error was detected
in the structure. Surface residue 537 was found to be an Asp/Asn
instead of a Gly predicted by the sequence of the native enzyme. The
nucleotide sequence of the recombinant DNA identified the residue as an
Asp. The stereochemistry of the final model was analyzed by PROCHECK
(27): 91% of the residues were found to lie in the most favorable
regions of the Ramachandran plot and 8.6% in the additional allowed
regions. Only two residues (Glu344 and Phe250)
were found in a generously allowed region. A schematic representation of the enzyme is shown in Fig. 1. The overall B-factor for the protein
is 16.4 Å2, whereas it is 15.9 Å2 and 16.5 Å2 for the main chain and the side chain atoms,
respectively. The B-factors of the C
atoms are plotted in Fig. 2.
The active site is shown in Fig. 3 as an example of the quality of the
1
(2Fo-Fc) electron density.
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Coordinates--
Coordinates have been deposited at the Protein
Data Bank (accession code 1G5A).
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RESULTS AND DISCUSSION |
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Description of the Structure--
The single polypeptide chain
(628 amino acid residues) is folded into a tertiary structure with five
domains named N, A, B, B', and C (Fig.
1). Residues 1-90 comprise the all
-helical N-domain. It contains six amphiphilic helices that we have
chosen to name nh1 to nh6. The helices consist of the residues
Pro2-Leu12,
Thr16-Lys25,
Ser26-Pro41,
Pro41-Gly52,
Leu57-Arg75, and
Ser77-Asn88 as defined by the Kabsch-Sander
algorithm (28) in the program PROCHECK (27). No known structures or
domains were found to be similar to the AS N-domain in a database
search with the DALI (29) server. Two helices from the N-domain (nh4
and nh5) are packed against two helices (h3 and h4) from the central
(
/
)8-barrel (domain A) forming a four-helix bundle.
The interface between the helices in the bundle is almost entirely
hydrophobic, and no solvent molecules are located in the interface.
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Domain A (residues 98-184, 261-395, and 461-550) is made up of eight
alternating -sheets (e1-e8) and
-helices (h1-h8) giving the
catalytic core (the well characterized (
/
)8-barrel)
common to the GH Family 13 (Figs. 1 and 4). A characteristic of the
(
/
)8-barrel enzymes is that the loop region
connecting strands to helices (labeled loop1 to loop8) are much longer
on average than those connecting helices to strands. In particular AS
has two loops (loop3 and loop7) that are so long that they constitute
the separate domains B and B'. The positions of the secondary
structural elements within the primary structure of domain A are shown
in Fig. 4.
Domain B (residues 185-260) contains two short antiparallel
-sheets. The inner sheet (relative to the barrel) is formed by two
strands (residues 187-190 and 253-256) and the outer sheet is formed
by three strands (residues 211-213, 237-240, and 245-248) flanked by
two
-helices (residues 193-201 and 216-222) (Figs. 1 and 4). A
B-domain is found in many
-amylases. In TAKA-amylase the main
structural feature is a short three-stranded antiparallel
-sheet.
There are also
-amylases that do not have a B domain. For example,
barley
-amylase (30) has a short hairpin at this position.
Domain B' (residues 395-460) starts with two -helices (residues
400-407 and 410-422) (Figs. 1 and 4). They are followed by a short
-sheet (residues 433-436 and 446-449) where the strands are
separated by a hairpin-like stretch of amino acid residues. A short
-helix (residues 451-456) terminates the domain. The domain starts
immediately after two catalytically important residues (His392 and Asp393, see below) found in all
related enzymes.
Domain C is an eight-stranded -sandwich found C-terminal to the
(
/
)8-barrel (residues 555-628). A C domain is found
in other
-amylases, for example TAKA-amylase and barley
-amylase. Several of these domains are found in the CGTases, but so far the
functional role of the C domain is unknown.
Although the complete AS sequence contains six cysteine residues no disulfide bridges are found in the structure. Some of the cysteines are exposed on the surface, but no tendency to multimerization has been reported.
The C displacement parameters (B-factors) are plotted in Fig.
2. The enzyme displays low overall
thermal vibration with a mean B-factor for all atoms of 16.4 Å2. The plot shows that especially the region 250-400
(from the start of h3 to the beginning of the B' domain) has low
displacement parameters and that the regions of the molecule with the
highest displacement parameters are localized far from the substrate
binding pocket.
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Relation to Family 13 Enzymes--
A comparison of the full-length
enzyme with known protein structures using the DALI server (29) showed
that the glycoside hydrolase Family 13 exo-acting enzyme
oligo-1,6-glucosidase (31) had the highest similarity to AS. A total of
458 C atoms could be superimposed with an rms of 2.7 Å. The
superimposable residues are almost all found in the A and C domains.
The structural similarity to Family 13
-amylases is also high. In
particular, the TAKA-amylase-acarbose complex (32) had 368 superimposable C
atoms with a rms of 2.8 Å. A structural-based
sequence alignment between TAKA-amylase, AS, and oligo-1,6-glucosidase
starting after the unique N-domain is shown in Fig. 4. The boxed
sequence patches represents regions of genuine structural similarity.
Because of the high structural similarity the alignment can be used to
propose AS-substrate interactions from enzyme-substrate investigations
performed on related enzymes.
Active Site Architecture--
The general acid residue
Glu328 and the nucleophile Asp286 have been
identified using conventional sequence alignment (9) and mutational
studies (10). These results are supported by the structural alignment
found in Fig. 4, which shows that the C positions of the two
residues coincides with catalytic residues from both TAKA-amylase and
oligo-1,6-glucosidase. Asp286 and Glu328 are
found at the tips of
-sheets 4 and 5 in the
(
/
)8-barrel of AS (Fig. 1), as required for Family 13 members. The distance between Asp286 C
and
Glu328 C
is 5.4 Å in accordance with AS being an
-retaining enzyme (14). A Tris molecule is bound at the active site
(Fig. 3) with a short hydrogen bond (2.6 Å) between O
2 of Asp286 and one of the Tris oxygens and
several hydrogen bonds to surrounding amino acid side chains
(Asp144, His187, Glu328, and
Arg509). Tris has previously been found to be a very good
probe for the active site of
-amylases (33).
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The TAKA-amylase-acarbose complex (32) has identified a number of
enzyme-substrate active site interactions. Around the 1 subsite the
following interactions are reported (conserved residues at an
equivalent position in AS given in parentheses): The nucleophile
Asp206 O
2 (Asp286) is forming a hydrogen
bond to the O6 of the I-ring of the modified acarbose (Fig. 5).
His122 (His187) also forms a hydrogen bond to
O6I. Arg204 (Arg284) forms a salt-bridge to the
O
1 of the nucleophile and is very important for the correct
positioning of the nucleophile. Arg204 (Arg284)
has an additional weak hydrogen bond to O2I.
Glu230 (Glu328) is the general acid/base. It
forms a hydrogen bond to the acarbose N. Asp297
(Asp393) O
1 and O
2 forms hydrogen bonds to
O2I and O3I respectively. Tyr82
(Tyr147) provides an important stacking platform for the
substrate ring at the
1 position. Finally His392
(His296) forms a short hydrogen bond to the hydroxyl O of
Tyr82 (Tyr147) an interaction suggested to be
pivotal for the positioning of the stacking platform (32). All of these
residues can be found at identical C
positions in
TAKA-amylase, AS, and oligo-1,6-glucosidase (Figs. 4 and 5).
As seen in Fig. 5, the side chains of these residues are found in
identical spatial positions as well. Thus
-amylases and AS have very
similar active site architecture with respect to the immediate
surroundings of the scissile bond (subsite
1). This is in agreement
with the general mechanism outlined in Scheme 1. The mechanism for the
formation of the covalent intermediate is similar. But how does AS
ensure specificity for sucrose as the first substrate, and how does AS
prevent water from being the second substrate? These questions can be
addressed by studying the superposition of AS and TAKA-amylase at the
other subsites mapped out by the TAKA-amylase-acarbose complex.
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Specificity for Sucrose as the First Substrate--
TAKA-amylase
is an endo-acting enzyme. It has a total of six specific binding sites
for linked -(1
4)-glucosyl moieties. The TAKA-amylase residues
reported to be involved in enzyme-substrate contacts in subsites +1 and
+2 are not structurally conserved in AS. TAKA-amylase
His210 in subsite +1 donating a hydrogen bond from N
2 to
O5J is a Phe in AS, whereas TAKA-amylase Lys209 with
hydrogen bonds to the modified acarbose hydroxyls OK2 and OK3 is an Ala
in AS. This suggests that the +1 subsite in AS is modified to
accommodate specificity for the furanosyl ring of sucrose.
The TAKA-amylase-2 subsite has been completely disrupted in AS.
Asp144 from loop2 forms a salt bridge with
Arg509 and thus occupies the subsite. An equivalent salt
bridge is observed in the exo-acting oligo-1,6-glucosidase. An Ala and
an Asp are found in these positions in TAKA-amylase. The salt bridges
gives the active site a pocket topology in AS and
oligo-1,6-glucosidase, in contrast to the cleft observed in
TAKA-amylase. The result of the pocket topology is an exo-acting
enzyme. The -amylase cleft is closed by residues from domain B,
domain B', and loop2. The bottom of the pocket is quite thin-walled
with a solvent accessible dent in the protein surface right behind the
Asp144-Arg509 salt bridge. Without this
blockage the active site topology would be a tunnel. In conclusion, the
assumed furanosyl specificity at the +1 site and the salt bridge
creates the sucrose specificity in AS.
Oligosaccharides as Second Substrates--
The pocket topology in
AS greatly reduces the solvent accessibility to the active site. When
examining the AS structure superimposed with the TAKA-amylase-acarbose
structure it can be seen that the pocket includes the 1 and +1
subsites. The superposition also suggests that Phe250 is
sandwiching the I-ring of the modified acarbose at the
1 subsite with
Tyr147 in AS. TAKA-amylase has a Gly at this position,
oligo-1,6-glucosidase also have a Phe. This could implicate a more
stable covalent intermediate in AS and oligo-1,6-glucosidase compared
with
-amylases. This in turns could reflect the reduced
accessibility of the active site. The intermediate simply has to exist
long enough for the fructose to leave the active site and the second
substrate (oligosaccharide or water) to enter.
The pyranosyl ring bound at the TAKA-amylase +2 subsite can just
be seen in the surface plot (Fig. 6).
Compared with the exo-acting hydrolase oligo-1,6-glucosidase the pocket
of AS is very narrow leaving little room for water to enter when an
oligosaccharide such as elongated acarbose is in the pocket. In the
superimposition of acarbose into the AS structure the modified acarbose
molecule fills the pocket almost completely. The surface area of the
enzyme around the pocket-entrance is however quite open (Fig. 6). In fact several ravines in the surface leads to the pocket. Hence, it
could be speculated that the growing glucan polymer is embedded in one
ravine, whereas sucrose/fructose approaches/leaves the active site
through another ravine. An architecture like this with a number of
remote glucose binding subsites (securing a high "effective"
concentration of the oligosaccharide chain) could be responsible for
the transferase rather than hydrolase activity of AS. The unique AS
domain-B' is involved in the formation of these ravines (Fig. 6,
dark gray surface). Because of the close proximity to the
active site and the high content of aromatic residues (7 Phe, 3 Tyr,
and 1 Trp out of 54 residues) it is tempting to propose that the
B'-domain is essential for the binding of the growing glucan polymer.
However, this hypothesis has to be tested by experiments involving
complex formation with different oligosaccharides.
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Calcium Independence--
No calcium ions were found in the
structure. This also makes AS more similar to oligo-1,6-glucosidase
than to TAKA-amylase. However, most of the Ca2+ site
arrangement found in many amylases is conserved. In TAKA-amylase a
calcium ion is hepta-coordinated by oxygen atoms from
Asp175 (O1 and O
2), Asn121 (O
1),
Glu162 (backbone O), His210 (backbone O), and
three water molecules. For both AS and oligo-1,6-glucosidase the
calcium ion found in
-amylases is replaced with a presumable protonated lysine N
(Lys293 in AS and Lys206
in oligo-1,6-glucosidase). The two side chains involved in calcium binding (Asn121 and Asp175 in TAKA-amylase) are
structurally conserved (Asn186 and Asp256) in
AS. However, only Asp256 O
2 is within hydrogen bonding
distance of the TAKA-amylase Ca2+ site. One of the backbone
interactions (O from His210 in TAKA-amylase) is also
conserved in AS (Phe290), but the change in side chain
disables the interaction between the "calcium" site and the subsite
+1 described for TAKA-amylase (32). The last of the three hydrogen
bonds found for Lys293 N
comes from a water molecule.
The lack of calcium has also been observed in neopullulanase (34) and
maltogenic
-amylase (35).
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ACKNOWLEDGEMENTS |
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We thank L. Jacobsen for help with the crystallization experiments and V. Stojanoff at BM 14 ESRF for help with the MAD data collection and processing.
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FOOTNOTES |
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* This work was supported by the EU biotechnology project Alpha-Glucan Active Designer Enzymes (AGADE, BI04-CT98-0022) and the Danish Synchrotron User Center (DANSYNC).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 1G5A) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Dept. of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
To whom correspondence should be addressed: Tel.: 45 35 32 02 80; Fax: 45 35 32 02 99; E-mail: gajhede@psg.ki.ku.dk.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M010998200
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ABBREVIATIONS |
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The abbreviations used are: AS, amylosucrase; GH, glycoside hydrolase; CGTase, cyclodextrin glucanotransferase; rms, root mean squared; MAD, multiple wavelength anomalous dispersion.
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