Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes

Article abstract of DOI:10.1073/pnas.1007531107

Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (β/α) ₈-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a "U"-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product.

Full text of DOI:10.1073/pnas.1007531107

Crystal structure of a 117 kDa glucansucrase
fragment provides insight into evolution and
product specificity of GH70 enzymes
Andreja Vujičić-Žagara, Tjaard Pijninga, Slavko Kraljb,2, Cesar A. Lópeza, Wieger Eeuwemab,
Lubbert Dijkhuizenb, and Bauke W. Dijkstraa,1
aLaboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; and bDepartment of Microbiology,
University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved October 20, 2010 (received for review May 30, 2010)
Glucansucrases are large enzymes belonging to glycoside hydro-
lase family 70, which catalyze the cleavage of sucrose into fructose
and glucose, with the concomitant transfer of the glucose residue
to a growing α-glucan polymer. Among others, plaque-forming oral
bacteria secrete these enzymes to produce α-glucans, which facil-
itate the adhesion of the bacteria to the tooth enamel. We deter-
mined the crystal structure of a fully active, 1,031-residue fragment
encompassing the catalytic and C-terminal domains of GTF180 from
Lactobacillus reuteri 180, both in the native state, and in complexes
with sucrose and maltose. These structures show that the enzyme
has an α-amylase-like ðβ∕αÞ8-barrel catalytic domain that is circu-
larly permuted compared to the catalytic domains of members
of glycoside hydrolase families 13 and 77, which belong to the
same GH-H superfamily. In contrast to previous suggestions, the
enzyme has only one active site and one nucleophilic residue.
Surprisingly, in GTF180 the peptide chain follows a “U”-path, such
that four of the five domains are made up from discontiguous
N- and C-terminal stretches of the peptide chain. Finally, the struc-
tures give insight into the factors that determine the different link-
age types in the polymeric product.
inal putative glucan-binding domain flanking the central catalytic
domain (7).
Because of the similarity of glucansucrases to GH13 α-amy-
lases and GH77 amylomaltases, glucansucrases have been pro-
posed to use an α-retaining double displacement mechanism
(Fig. S1A; 7, 11–15). In the first step, the αð1 → 2Þ-glycosidic
bond of sucrose is cleaved, fructose is released, and a glucosyl-
enzyme intermediate is formed in which the glucosyl unit is
covalently attached to the catalytic nucleophile via a β-glycosidic
linkage. In the second step, the covalently bound glucosyl moiety
is transferred to the accepting nonreducing end sugar of a grow-
ing glucan chain, with reformation of the α-glycosidic bond. In
addition, the glucosyl moiety may be transferred to a low mole-
cular mass acceptor substrate such as maltose or isomaltose, or
to a water molecule. An alternative mechanism proposes that the
acceptor is the reducing end of the growing polysaccharide chain
(Fig. S1B). This mechanism requires either two nearby catalytic
sites or one catalytic site with two nucleophilic residues that both
form covalent glucosyl-enzyme intermediates, and that alternat-
ingly transfer their glucosyl moiety to the C1 atom of the other
covalently bound glucose, freeing that nucleophile for a next
reaction cycle (16). In the absence of a three-dimensional struc-
ture of a GH70 enzyme the precise catalytic mechanism has
remained controversial.
Here we describe the crystal structure of a fully active GH70
glucansucrase of Lactobacillus reuteri 180, encompassing the cat-
alytic and C-terminal domains, but not its ∼740 residues N-term-
inal domain (17). This 117 kDa GTF180-ΔN (residues 742–1,772)
produces a high molecular mass (∼36 MDa) glucose polymer,
with both αð1 → 6Þ and αð1 → 3Þ glucosidic linkages (18). Crystal
structures with bound sucrose (substrate) and maltose (acceptor)
combined with site-directed mutagenesis experiments showed that
GH70 glucansucrases possess only one active site and have only
one nucleophilic residue. These results provide a solid basis for
structure-based inhibitor design, and could facilitate the search
for unique specific anticaries drugs. Surprisingly, the GTF180
peptide chain follows a “U”-path, such that four of its five domains
are made up from two, discontiguous N- and C-terminal stretches
of the peptide chain, respectively.
crystal structure complexes ∣ exopolysaccharide ∣ Lactobacillus reuteri ∣
dental caries
ental infections such as tooth decay and periodontal diseases
D
are amongst the most common bacterial infections in
humans. The causal connection between dental caries and the
intake of dietary sugar is well established (1). Fermentation of
carbohydrates by plaque-forming oral bacteria produces acids,
which cause the dissolution of calcium phosphate in the tooth
enamel (2). In addition, dietary sucrose serves as a substrate for
bacterial glucansucrases, which produce α-glucan polysaccharides
that facilitate biofilm formation and significantly enhance the ad-
hesion of bacteria to the tooth enamel (3–5). Therefore, glucan-
sucrases have been proposed as potential targets for anticaries
drugs. Although a wide range of inhibitory substances has been
investigated, none has yet been discovered that is sufficiently
specific for glucansucrases and that does not have adverse side
effects, e.g., by inhibiting other carbohydrate hydrolyzing en-
zymes in the oral cavity (6).
Intestinal lactic acid bacteria also secrete glucansucrases, and
to date over 80 different glucansucrases are known (7, 8).
Glucansucrase enzymes have been classified in glycoside hydro-
lase family GH70 (9) on the basis of four catalytically important
conserved sequence motifs that are similar to those of members
of glycoside hydrolase families GH13 and GH77, but that occur
in a different order. This observation led to the proposal that
glucansucrases belong to the α-amylase superfamily (or GH-H
clan), but have a circularly permuted ðβ∕αÞ8-barrel as catalytic
domain (10). However, glucansucrases are much larger enzymes
(∼1;600–1;800 amino acid residues) than their GH13 and GH77
relatives (∼500–600 amino acids), and they contain an N-terminal
domain of variable length and unknown function and a C-term-
Author contributions: A.V.-Z., S.K., L.D., and B.W.D. designed research; A.V.-Z., T.P., S.K.,
C.A.L., and W.E. performed research; A.V.-Z., T.P., and S.K. analyzed data; and A.V.-Z.,
S.K., and B.W.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 3KLK, 3HZ3, and 3KLL).
1To whom correspondence should be addressed. E-mail: b.w.dijkstra@rug.nl.
2Present address: Genencor—A Danisco Division, Archimedesweg 30, 2333 CN Leiden,
The Netherlands.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1007531107/-/DCSupplemental.
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Results
Domains IVand V. Domain IV is positioned between domains B and
V (Fig. 1A). Domain IV has several interactions with the B do-
main, but very few with domain V. Domain IV has no structural
similarity to other proteins in the Protein Data Bank (DALI
Z-scores < 2.5). The function of domain IV is obscure, beyond
providing a linker between the catalytic core of the enzyme and
domain V. Domain V contains the first 47 and the last 114 visible
residues of the GTF180-ΔN enzyme. Both polypeptide segments
contain ∼20 amino acids long sequence repeats known as putative
cell wall binding repeats (19). Each repeat defines a β-hairpin fol-
lowed by a loop connecting consecutive hairpins (Fig. 1A, domain
V). Two of such repeats are found at the N- and five at the C-term-
inal end of the GTF180-ΔN sequence. Although these sequence
repeats were proposed to be involved in glucan binding (7, 8),
soaking of GTF180 crystals with a variety of sugars (glucose, iso-
maltose, panose, and nigerotriose) did not reveal any sugar bind-
ing to this domain. Domain V shows sequence and structural
similarity to the choline-binding domain of Streptoccocus pneumo-
niae LytA [(20); 29% identity; rmsd of 2.1 Å for 106 Cα atoms],
but choline-binding could neither be demonstrated.
Overall Structure. Lactobacillus reuteri 180 GTF180-ΔN, eluci-
dated at 1.7 Å resolution, has an elongated shape of dimensions
150 × 60 × 60 Å3 and folds into five distinct, linearly arranged do-
mains (Fig. 1A; for crystallographic details see Tables S1 and S2).
Three of these domains structurally align with the A, B, and C
domains of family GH13 α-amylases and therefore were named
accordingly. The other two domains do not show structural simi-
larity to domains occurring in the GH13 family and were thus
named IVand V. The five domains are not arranged consecutively
along the polypeptide chain. From the N to the C terminus, the
polypeptide chain contributes first to domain V, then to domain
IV, next to B, then to A and finally to domain C, and then returns
back to domain V via domains A, B, and IV, respectively. The
polypeptide chain thus follows a “U”-path (Fig. 1B).
The A, B, and C Domains. Domain A is the catalytic domain; it
contains a ðβ∕αÞ8-barrel, which is indeed “circularly permuted”
(10); the domain starts with the helix that is equivalent to the
α3 helix of the GH13 ðβ∕αÞ8-barrel, while the last strand is
equivalent to strand β3 of the GH13 enzymes (α3, β4α4, …,
β8α8, β1α1, β2α2, β3 arrangement, Fig. S2). Domain B is inserted
between helix α1 and strand β8 of the ðβ∕αÞ -barrel. The domain
contributes several amino acid residues to8the substrate donor
and acceptor binding sites (see below). Because of the equiva-
lence of the GH70 β8/α1 and GH13 β3/α3 secondary structure
elements domain B occupies the same relative position in both
enzyme families (Fig. S2). A single heptacoordinated Ca2þ ion
is bound in the interface between domains A and B (Fig. 2
and Video S1). Treatment of the enzyme with the Ca2þ-chelating
agent EDTA (ethylenediaminetetraacetic acid) resulted in ∼40%
lower activity (see Table S3), which might be due, among others,
to a compromised binding of the substrate/acceptor by N1029
(see below). Domain C (residues 1,238–1,376) forms the bottom
of the “U.” Even though most α-amylase superfamily enzymes
have this domain its precise function is unclear.
Sucrose Binding. A 2.2 Å resolution structure of the inactive
D1025N mutant protein with bound sucrose substrate showed
that sucrose binds in the active site, with its glucosyl moiety in
subsite −1 and its fructosyl part in subsite þ1 [Fig. 2A and
Video S1; for a definition of the subsites, see Davies et al. (21)].
Six of the seven strictly conserved GH13 active-site residues (22)
are present in GH70 GTFs, making similar interactions with the
−1 glucose as in GH13 enzymes [Fig. 2A; (14, 23)]. Among these
strictly conserved residues are the putative catalytic acid/base
(E1063 in GTF180) and nucleophile (D1025; mutated to N in
the sucrose-bound structure), which, in agreement with their
proposed role, are oriented towards the glycosidic oxygen and
anomeric carbon of the −1 glucosyl moiety, respectively. Mutat-
ing these residues virtually inactivates the enzyme (Table S4). The
putative transition state stabilizer D1136 is at 2.8 Å from O2, and
may reduce the electronegativity of O2 (14, 24). The crucial role
in catalysis of D1136 is affirmed by the drastic decrease in activity
of the GTF180-ΔN D1136N-mutant (Table S4). The conserved
H1135 contacts the glucosyl O2 and O3 hydroxyl groups, and
R1023 hydrogen bonds the O2 hydroxyl group, similar to the
equivalent residues in GH13 enzymes. The sixth conserved resi-
due, D1504, is not located in the catalytic center, but it makes a
H-bond with the hydroxyl group of the conserved Y1465, which
has hydrophobic stacking interactions with the sucrose glucosyl
moiety in subsite −1.
A
B
The seventh conserved residue in GH13 enzymes is a histidine
that hydrogen bonds to the O6 hydroxyl group of the −1 glucose
(22). In GTF180 this residue is Q1509, which fulfils, however, the
same function of hydrogen bonding to O6. In subsite þ1 the β-D-
fructosyl moiety makes hydrogen bonds with E1063, W1065,
N1029, and Q1140 (Fig. 2A).
Importantly, no evidence was obtained for a second sucrose-
binding site near the active site, as required for the two catalytic-
site/double-nucleophile mechanism (16). A second sucrose mole-
cule is bound near W1531, approximately 25 Å from the catalytic
site (Fig. S3A), but lack of conservation of the binding residues
suggests that sucrose binding at this distant site is not of general
significance for the GH70 family. Moreover, a W1531S mutation
had no effect on the product size (Fig. S4).
Maltose Binding. The disaccharide maltose is a good acceptor
substrate (25–27). A structure of native GTF180-ΔN crystals with
bound maltose (2.0 Å resolution) revealed four maltose-binding
sites, M1, M2, M3, and M4 (see Tables S1 and S2 for crystallo-
graphic statistics). The M2–M4 binding site residues are not
conserved in other GH70 family enzymes and therefore seem
Fig. 1. Overall structure of L. reuteri 180 GTF180-ΔN. (A) Crystal structure of
GTF180-ΔN, the N- and the C-terminal ends of the polypeptide chain are in-
dicated, the Ca2þ ion is shown as a magenta sphere; (B) Schematic presenta-
tion of the “U-shaped” course of the polypeptide chain. Domains A, B, C, IV,
and Vare coloured in blue, green, magenta, yellow, and red, respectively, with
dark and light colors for the N- and C-terminal stretches of the peptide chain.
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Fig. 2. Stereo views of sucrose and maltose binding
in the active site of GTF180(D1025N)-ΔN. (A) Sucrose
(white stick representation) bound at subsites −1 and
þ1 in the D1025N mutant. Residues of the 980–984
loop are depicted as a transparent cartoon for clarity.
Strictly conserved residues in the α-amylase super-
family are labeled in bold, except for D1504 which
is not shown; (B) Maltose M1 (yellow stick presenta-
tion) bound in subsites þ1 and þ2. Maltose M2
bound at subsites þ2 and þ3 is also shown. The glu-
cosyl moiety of the sucrose from the GTF180-ΔN-
sucrose complex (white stick representation) is super-
imposed. Leu938 in front of the þ1 glucosyl residue
was omitted for clarity. Residues from domains A and
B are colored blue and green, respectively. The Ca2þ
ion is shown as a magenta sphere, water molecules
are shown as smaller red spheres. Hydrogen bonding
interactions are shown as black dashed lines.
of no importance to the GH70 family in general. A description of
the M3 and M4 binding sites can be found in the SI Text.
Maltose M1 is bound at subsites þ1 and þ2. The þ1 glucosyl
moiety of M1 is sandwiched between W1065 on one side and
L938 and L981 on the other side (Fig. 2B and Video S2). The
þ1 C6 hydroxyl group is at hydrogen bonding distance from
the catalytic acid/base, E1063, in a productive position to attack
a covalently bound enzyme-glucosyl-intermediate. The þ1 C4
hydroxyl group makes a direct, short hydrogen bond (2.6 Å) with
N1029. The þ2 glucose residue has van der Waals interactions
with W1065 (Fig. 2B). The disaccharide shows no deviations
from the low energy maltose torsion angles and also the hydrogen
bond normally present between the C2 and C3 hydroxyl groups of
adjacent αð1 → 4Þ-linked glucose molecules is formed. Interest-
ingly, maltose M2 binds near the active site (Fig. 2B). However,
the M2 binding mode is rather aspecific (via H-bonding interac-
tions with the þ1 glucose, and water-mediated hydrogen bonds
with the protein) and therefore unlikely to mimic a bona fide
acceptor substrate.
docking studies in a model of the covalent glucosyl-GTF180-ΔN
intermediate (in subsite −1), which was made based on the
structure of the glucosyl-intermediate of Neisseria polysaccharea
amylosucrase [PDB code 1S46; (23)]. Like maltose, isomaltose
(Glc-αð1 → 6Þ-Glc) binds with its nonreducing glucose residue
in subsite þ1; its O6 hydroxyl group points towards the position
of the C1 atom of the covalently bound sugar in subsite −1
(Fig. 3A). The reducing end glucose binds in subsite þ2, and
has stacking interactions with W1065. The isomaltose binding
mode shows how an αð1 → 6Þ-linked glucan can be extended by
another glucose residue via an αð1 → 6Þ-linkage.
A different mode of binding was shown by isomaltotriose
(Glc-αð1 → 6Þ-Glc-αð1 → 6Þ-Glc), which binds in subsites þII’,
þ1 and þII” (Fig. 3B). The O3 atom of the central, þ1 glucose
residue is directed towards the C1 atom of the glucosyl-enzyme
intermediate. This binding mode illustrates how an αð1 → 3Þ
branch point can be introduced in the glucan chain. In principle,
isomaltose could also bind in subsites þ1 and þII” with O3 point-
ing towards the C1 atom at subsite −1. This binding mode would
allow elongation of a linear αð1 → 6Þ-glucan chain with an
αð1 → 3Þ-linked glucose.
Analysis of Products Synthesized by GTF180-ΔN. Product analysis
of GTF180-ΔN incubated with both sucrose and maltose present
Docking of the disaccharide nigerose (Glc-αð1 → 3Þ-Glc)
in a 1∶1 ratio revealed that ∼42% of maltose was converted into resulted in the þ1 glucose predominantly being bound with its
the trisaccharide panose (Glc-αð1 → 6Þ-Glc-αð1 → 4Þ-Glc; Glc is O6 hydroxyl group pointing towards the C1 atom of the glucosyl-
glucose) and ∼11% into the tetrasaccharide αð1 → 6Þ-panose enzyme intermediate (Fig. 3C), while the reducing end glucose oc-
(Glc-αð1 → 6Þ-panose) (Fig. S5 and Ta bl e S 5, for details on cupies subsite þ2 (Fig. 3C). This binding mode shows how a glu-
product analysis see SI Text). These results show that the glucosyl cosyl moiety can be added to the O6 hydroxyl group of an acceptor
moiety of sucrose is transferred to the nonreducing end of maltose saccharide of which the terminal nonreducing sugar is linked via
and panose. Transfer to the nonreducing end is also in agreement
with other studies (13, 15) as well as with the structure of GTF180-
ΔN with a bound maltose, which shows that maltose binds adjacent
to subsite −1 with its nonreducing end C6 hydroxyl group pointing
towards the catalytic center. Thus, the O6 atom can act as the
nucleophile in the second reaction step, leading to the extension
of an α-glucan at its nonreducing end by one glucose residue.
an αð1 → 3Þ linkage. The docking of nigerose did not result in a
binding mode allowing elongation at the O3 hydroxyl group.
Discussion
Discontiguous Domain Arrangement. Our results have revealed the
structural details of a GH70 glucansucrase. In contrast to the
previously proposed linear arrangement of domains along the
polypeptide chain (7, 8), the structure of L. reuteri 180 GTF180-
ΔN shows that four of the five domains are built up from discon-
tiguous parts of the polypeptide chain. Only the C-domain is
Binding Modes of Natural Acceptor Oligosaccharides. To analyze
the acceptor specificity of the enzyme we carried out automatic
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Fig. 3. Docking of isomaltose (A), isomaltotriose (B), and nigerose (C) in the active site of a modeled glucosyl-GTF180 intermediate. For modelling and docking
procedure see SI text. The covalently bound glucosyl moiety is shown in white stick representation, isomaltose, nigerose, and isomaltotriose are shown in
yellow stick representation. Dashed lines show hydrogen bonding distances of less than 3.5 Å, solid yellow lines show where the chain may extend; subsites
are numbered with Arabic and Roman white numerals. Domains A and B of GTF180 are shown in surface presentation and are coloured blue and green,
respectively.
formed from one continuous stretch of amino acids. Neverthe-
less, heterologous expression of the enzyme in Escherichia coli
is not problematic, showing that folding intermediates are stable
and that the N-terminal variable domain, which was absent in
our construct, is not needed for correct folding of the enzyme.
The structure confirms that, compared to GH13 α-amylases,
the catalytic ðβ∕αÞ8-barrel domain is circularly permuted as was
previously proposed (10).
The “permutation per duplication model” proposes a se-
quence of gene arrangements that could have led to a circularly
permuted gene via gene duplication, in-frame fusion, and partial
truncation (28, 29). In this way a GH70 glucansucrase precursor
consisting of a circularly permuted domain A, a discontiguous do-
main B, and a contiguous domain C could have arisen from the
GH13 α-amylase fold (Fig. 4). Insertion of this precursor into the
gene of domain IV, followed by insertion into the gene encoding
domains Vand N, could then have led to the present day protein.
Alternatively, domain IV may have inserted into the ancestor
α-amylase before the circular permutation occurred. In both
cases various intermediate forms must have existed during evolu-
tion. The requirement that such intermediate forms are stable
and should offer some form of advantage to the organism to pre-
vent removal from the gene pool, can explain why the number of
circular permutations observed in proteins is rather small (30).
the binding of longer oligosaccharides (see Fig. 2A and
Video S1). These residues are strictly conserved in GH70 family
enzymes, indicating that the binding of a single glucose residue in
subsite −1 is a general property of GH70 glucansucrases. In the
GH13 amylosucrase from N. polysaccharea, which produces an
αð1 → 4Þ-linked glucan polymer from sucrose, the glucose-bind-
ing pocket is also closed off, but by a salt-bridge between R509
and D144 (31).
Six residues that interact with the −1 glucose in GTF180-ΔN
are strictly conserved in GH70 and GH13 enzymes, and exhibit
comparable interactions with the −1 glucose residue (see Results).
In addition, Q1509 has a similar hydrogen bonding interaction
with the O6 hydroxyl group of the −1 glucose as the equivalent
His in the GH13 family. Thus, the active site of GTF180-ΔN is
very similar to those of GH13 family members. No space is avail-
able for a glucose covalently bound to D1136, as required for the
two-nucleophile mechanism proposed by Robyt et al. (16).
The similarity of the active sites of GH70 and GH13 enzymes
suggests a very similar reaction mechanism for the cleavage of the
α-glycosidic bond of sucrose and the formation of the β-glucosyl-
enzyme intermediate. Cleavage of the glycosidic bond of sucrose
is likely started by protonation of the glycosidic oxygen by E1063,
resulting in bond cleavage, release of fructose, and formation of a
partly planar oxocarbenium ion-like intermediate (12, 14, 24, 32).
Conceivably, D1136 pushes the −1 O2 atom towards the inter-
mediate position, while Q1509 holds the −1 glucose O6 atom.
Similar to GH13 enzymes, the C2-O2 bond of the −1 glucose re-
sidue is polarized by interactions of the O2 atom with H1135,
D1136, and R1023, which lowers the energy of the transition state
Active Site and Formation of the Covalent Glucosyl-Enzyme Intermedi-
ate. The active site of GTF180-ΔN is a pocket-shaped cavity, in
which the glucose moiety of sucrose binds (subsite −1). The cavity
is closed off by residues Q1140, N1411, and D1458, which prevent
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Fig. 4. Hypothetical evolutionary pathway based on the “permutation per duplication model” (28) leading to the unusual domain organization of GH70
family GTF180 starting from a putative GH13 α-amylase precursor. Sequence segments forming domains A, B, C, IV, and V are colored as before in blue, green,
magenta, yellow, and red, respectively.
(14, 22, 33, 34). The reaction is completed by nucleophilic attack Conclusions
of D1025 on the glucose C1 atom, which results in a covalent The crystal structures of GTF180-ΔN have revealed the molecu-
intermediate with the glucosyl moiety attached to D1025 via a
β-glycosidic linkage. Because the þ1 fructosyl moiety of sucrose
has fewer interactions with the enzyme than the −1 glucosyl moiety,
it can easily be released from the active site in order to allow the
reaction to proceed by binding the acceptor molecule at subsite þ1.
lar details of a GH70 enzyme and its interaction with sucrose and
the acceptor maltose. The data show that, in contrast to previous
proposals, only a single active site is present, and no space for
a second covalently bound glucose residue or glucan polymer
is available. This finding invalidates the proposal of a double ac-
tive-site/double-nucleophile insertion mechanism (16). Also the
conservation of key amino acids involved in substrate binding
and catalysis at subsite −1 in both GH13 and GH70 enzymes
confirms that the mechanism of glycosidic bond cleavage by
GH70 glucansucrases is similar to that of the α-amylase super-
family enzymes (14, 23).
The crystal structure of GTF180-ΔN with bound maltose
revealed how an acceptor substrate binds to receive a glucosyl
group at its O6 atom. Modelling showed that transfer of the glu-
cosyl group to the O3 atom requires an entirely different binding
mode (Fig. 3B).
Transfer of Glucose to the Acceptor Saccharide and Product Specifi-
city. The next step in the reaction mechanism is the transfer of
the covalently bound glucosyl residue to the acceptor saccharide.
GTF180-ΔN produces a branched dextran built up from different
lengths of αð1 → 6Þ-linked glucose residues, interconnected by
single αð1 → 3Þ-bridges. No consecutive αð1 → 3Þ glucosidic
bonds are present (35). The enzyme must thus be able to transfer
the glucosyl residue to either the O6 or the O3 hydroxyl group.
This requirement implies that the accepting glucose must be able
to bind in two different orientations, either with its O6 or with its
O3 hydroxyl group pointing towards the catalytic site. Our dock-
ing studies showed that these two binding modes are indeed pos-
sible (Fig. 3). At subsite þ2 the acceptor sugars are mostly bound
via aspecific stacking interactions with W1065; therefore the en-
zyme is not very particular about the specific glycosidic linkage
between the sugars in subsites þ1 and þ2, and can accommodate
αð1 → 3Þ, αð1 → 4Þ, or αð1 → 6Þ-linked glucoses. In all cases the
accepting O6 hydroxyl group will be near the C1 atom of the
covalent intermediate.
In case the O3 hydroxyl group is the acceptor, a different bind-
ing mode of the acceptor is needed, as exemplified by the docking
results with isomaltotriose (Fig. 3B). In this case, the reducing
end glucose binds in subsite II” rather than subsite þ2, and
the nonreducing end extends towards subsite þII’. In this binding
mode an αð1 → 3Þ branch point is introduced in the glucan chain.
Although our docking studies with isomaltose did not provide evi-
dence, it can be envisaged that occasionally also a terminal
αð1 → 6Þ-linked glucan may bind in subsites þ1 and þII”, with
O3 pointing into the active site. Such a binding mode would allow
linear glucan chain elongation with an αð1 → 3Þ-linked glucose.
The docking results for both isomaltose and nigerose suggest
that the most favorable glucan conformer is the one allowing
formation of the αð1 → 6Þ linkage. Indeed, the majority (69%)
of the glycosidic bonds in the α-D-glucan synthesized by
GTF180 from sucrose are αð1 → 6Þ-bonds and only about 31 %
are αð1 → 3Þ-bonds (18, 35).
Finally, in view of the conservation of the residues involved in
subsite −1 in enzymes from families GH13, GH70, and GH77, the
design of specific anticaries glucansucrase-inhibitors is predicted
to require the exploitation of features of the enzymes more
remote from subsite −1.
Experimental Procedures
Cloning, Mutagenesis, Purification, Crystallization, and Soaking
Experiments. For cloning and mutagenesis of gtf180 see SI text.
Purification and crystallization of native L. reuteri GTF180-ΔN
were performed as described previously (17). Selenomethionine
(SeMet) labeled GTF180-ΔN was produced as formerly reported
(36). Both the D1025N mutant and SeMet labeled GTF180-ΔN
crystallized in the same conditions as the native protein. Crystals
of the D1025N mutant were soaked for 30 min in mother liquor
(25% ðw∕vÞ PEG 3350, 50 mM NaCl, 50 mM 1,3-bis[tris(hydro-
xymethyl)methylamino]propane HCl, pH 6.0, 2 mM CaCl2)
containing 25 mM sucrose, and cryoprotected in mother liquor
containing 35% ðw∕vÞ PEG 3350 and 25 mM sucrose. For mal-
tose soaking studies a crystal of SeMet-GTF180-ΔN was trans-
ferred first to mother liquor supplemented with 25 mM maltose
(15 min), and then soaked for 2 h in mother liquor containing
250 mM maltose. Crystals were cryoprotected in 35% ðw∕vÞ
PEG 3350 and 250 mM maltose.
21410
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www.pnas.org/cgi/doi/10.1073/pnas.1007531107
Vujičić-Žagar et al.

Data Collection. All datasets were collected at 100 K. Multiple
wavelength anomalous dispersion (MAD) and “native” datasets
were collected at beam line ID29 at the European Synchrotron
Radiation Facility (ESRF), Grenoble, France and processed
using MOSFLM and SCALA (37). The “sucrose” and “maltose”
datasets were collected at beam lines BM16, ESRF, Grenoble,
France, and BW7A, European Molecular Biology Laboratory
(EMBL) outstation at the Deutsches Elektronen-Synchrotron,
Hamburg, Germany, respectively. The latter two datasets were
processed using the XDS package and SCALA (37, 38). All crys-
tals belonged to space group P1, with one molecule per unit cell
and 52% solvent content. The presence of one molecule per unit
cell is in agreement with gel filtration (Fig. S6) and dynamic light
scattering experiments (estimated molecular mass is 110 kDa
with 13.1% polydispersity), which indicate that the protein is
monomeric in solution. For data collection and processing statis-
tics see Table S1.
Model building and corrections were done using the program
COOT (42). The final native model consists of 1,006 amino acid
residues (residues 746–1,751). The first 4 (residues 742–745) and
the last 21 residues (residues 1,752–1,772) are invisible in the
electron density maps. Both sucrose and maltose were included
in the model in the final stages of the refinement. The resulting
structures were validated with MolProbity (43). For refinement
statistics see Table S2. Coordinates and structure factors have
been deposited with the Protein Data Bank (entries 3KLK,
3HZ3, 3KLL). Figures were created using PyMol (44).
Product Analysis. Products synthesized by GTF180-ΔN were ana-
lyzed as described in SI text.
Docking Studies. The automatic docking calculations were done
with the program AutoDock 4 (45). More details can be found
in SI text.
Structure Determination. The GTF180-ΔN structure was solved
with the program SOLVE/RESOLVE (39) using MAD data col-
lected around the Se edge. The experimental phase information
after RESOLVE was combined with native 1.7 Å data and used
for further automated model building and refinement using the
programs ARP/wARP (40) and REFMAC5 (41), respectively.
ACKNOWLEDGMENTS. We are grateful to staff scientists and local contacts
at beam lines ID29 and BM16 at the ESRF, Grenoble, France, and BW7A at
the EMBL-Hamburg outstation (DESY), Germany for their support during
data collection. The authors thank M. Tietema for construction of GTF180-
ΔN W1531S mutant and TLC analysis. This work was financially supported
by Senter Innovatiegerichte Onderzoeksprogramma’s, The Netherlands
(IGE01021).
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