Kineticsofdextran-independent a-(1
fi
3)-glucan synthesis
by Streptococcussobrinusglucosyltransferase I
Hideyuki Komatsu
1
, Yoshie Abe
1
, Kazuyuki Eguchi
1
, Hideki Matsuno
1
, Yu Matsuoka
1
, Takayuki
Sadakane
1
, Tetsuyoshi Inoue
2
, Kazuhiro Fukui
2
and Takao Kodama
1
1 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Japan
2 Department of Oral Microbiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Japan
Introduction
Water-insoluble glucan, which is mainly composed of
a-(1 fi 3)-glucan, enhances the colonization of cario-
genic bacteria and promotes the formation of dental
plaque on smooth tooth surfaces [1]. Two glucos-
yltransferases (GTFs) or glucansucrases (GTF-S and
GTF-I, EC 2.4.1.5) from mutans streptococci are
responsible for the production of this polysaccharide
[1,2]. GTF-S and GTF-I catalyze the synthesis of
water-soluble a-(1fi 6)-glucan and water-insoluble
a-(1 fi 3)-glucan, respectively. They share highly
homologous amino acid sequences and comprise
the N-terminal glucansucrase domain (GSd) and the
C-terminal glucan-binding domain (GBd) [3,4]. The
GSd catalyzes glucosyl transfer from sucrose to low
molecular mass acceptors such as water, isomaltose,
and maltose [5]. On the other hand, the GBd binds to
glucans such as dextran [6–8].
Unlike GTF-S, pre-existing dextran increases GTF-I
activity [9,10]. Although truncation of the GBd of
GTF-I by genetic engineering results in decreased
Keywords
enzyme kinetics; glucansucrase;
glucosyltransferase; mutans streptococci;
nigerooligosaccharide
Correspondence
H. Komatsu, Department of Bioscience and
Bioinformatics, Kyushu Institute of
Technology, Kawazu 680-4, Iizuka 820-8502,
Japan
Fax: +81 948 29 7801
Tel: +81 948 29 7845
E-mail: hide@bio.kyutech.ac.jp
(Received 3 September 2010, revised 18
November 2010, accepted 25 November
2010)
doi:10.1111/j.1742-4658.2010.07973.x
Glucosyltransferase (GTF)-I from cariogenic Streptococcussobrinus elon-
gates the a-(1fi 3)-linked glucose polymer branches on the primer dextran
bound to the C-terminal glucan-binding domain. We investigated the GTF-
I-catalyzed glucan synthesis reaction in the absence of the primer dextran.
The time course of saccharide production during dextran-independent glu-
can synthesis from sucrose was analyzed. Fructose and glucose were first
produced by the sucrose hydrolysis. Leucrose was subsequently produced,
followed by insoluble glucan [a-(1 fi 3)-linked glucose polymers] after a
lag phase. High levels of intermediate nigerooligosaccharide series accumu-
lation were characteristically not observed during the lag phase. The results
from the enzymatic activity of the acceptor reaction for the nigerooligo-
saccharide with a degree of polymerization of 2–6 and methyl a-
D-gluco-
pyranoside as a glucose analog indicate that the activity increased with an
increase in the degree of polymerization. The production of insoluble glu-
can was numerically simulated using the fourth-order Runge–Kutta
method with the kinetic parameters estimated from the enzyme assay. The
simulated time course provided a profile similar to that of experimental
data. These results define the relationship between the kinetic properties
of GTF-I and the time course of saccharide production. These results are
discussed with respect to a mechanism that underlies efficient glucan
synthesis.
Abbreviations
DP, degree of polymerization; GBd, glucan-binding domain; GS, glucan-binding domain-deficient glucosyltransferase-I; GSd, glucansucrase
domain; GSGB, glucosyltransferase-I containing a full-length glucan-binding domain; GTF, glucosyltransferase.
FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS 531
glucan synthesis [11–13], GBd-deficient GSd can still
synthesize a-(1fi3)-glucan from sucrose in the
absence of the pre-existing dextran (i.e. in a dextran-
independent manner) [14], albeit at a lower rate. This
suggests that the glucan synthesis reaction catalyzed by
the GSd is a basic function of GTF-I.
Although knowledge of the dextran-independent
reaction is important for a comprehensive understand-
ing of GTF-I function, its underlying mechanism
remains incompletely defined. In this respect, two con-
trasting mechanisms have been proposed for glucansuc-
rases. One mechanism is proposed by Robyt et al. from
pulse ⁄ chase experiments using [
14
C]sucrose [15,16]. This
mechanism involves processive elongation by which the
enzyme continuously transfers glucose residues to only
one acceptor molecule until the release of the final
product. Another mechanism is suggested by Mooser
et al. on the basis of the identification of the active site
and the similarity with established glycosidase mecha-
nisms [5,17]. The mechanism occurs via nonprocessive
elongation, whereby the enzyme releases the products
after each transfer of a glucose residue to the acceptor.
Steady-state kinetic studies have been performed for
other glucansucrases as well as fructansucrase [18–22].
In addition, the processes of insoluble glucan synthesis
from sucrose, investigated with high-performance anion
exchange chromatography analysis, are also reported
[23,24]. However, it is not clear how changes in the
intermediate oligosaccharides during the glucan syn-
thesis can be interpreted with respect to the kinetic
properties of enzymes. In other words, the relationship
between the enzyme kinetic properties and the process
of insoluble glucan synthesis remains unknown.
To address these issues, we analyzed the process of
dextran-independent insoluble glucan formation and
examined the enzyme kinetic properties of the nigero-
oligosaccharide acceptor reaction by using GBd-deficient
GTF-I (GS) and GTF-I containing a full-length GBd
(GSGB) from Streptococcussobrinus 6715 (serotype g)
(Fig. 1). A numerical simulation based on the enzyme
kinetic parameters is in good agreement with the time
course ofdextran-independent insoluble glucan forma-
tion. These results suggest that the dextran-independent
synthesis of insoluble glucan by GTF-I proceeds via
the nonprocessive elongation ofa-(1fi 3)-linked glu-
cose polymers (nigerooligosaccharides).
Results
Functional characterization of GSGB
We previously investigated the functional role of the
GBd in dextran-dependent a-(1fi3)-glucan synthesis
using GTF-I¢ (Asp85–Ile1256), which contains the GSd
and the 246 N-terminal residues of the GBd (approxi-
mately 50% of the GBd). The results indicate that the
GBd enhances a-(1fi 3)-branch formation on GBd-
bound dextran [14]. In the present study, we character-
ized newly prepared GSGB (Asp85–Asn1592) as an
enzyme possessing the full-length GBd (Fig. 1).
Figure 2A shows the dextran-dependent synthesis of
insoluble glucan as monitored by light scattering.
GSGB rapidly produced insoluble glucan in the pres-
ence of dextran (filled squares in Fig. 2A), whereas GS
did not (open squares in Fig. 2A). When glucosyl
transfer activity (initial velocity) was measured as a
function of dextran concentration, the activity of
GSGB was highly dependent on dextran (filled circles
in Fig. 2B); however, that of GS was nearly zero, inde-
pendently of dextran concentration (open circles in
Fig. 2B). On the other hand, there was no significant
difference in sucrose hydrolysis activity in the absence
of dextran between GS and GSGB. In addition, the
sucrose hydrolysis activities of GS and GSGB as a
function of sucrose concentration obeyed simple
Michaelis–Menten kinetics, with similar k
cat
and K
m
values (data not shown). The k
cat
and K
m
values were
as follows: GS, 11.4 ± 0.4 s
)1
and 0.37 ± 0.07 mm;
GSGB, 12.0 ± 0.4 s
)1
and 0.29 ± 0.04 mm. These
values are corroborated by our previous report [14].
Time course ofdextran-independent glucan
synthesis
We examined the kineticsof insoluble glucan synthesis
from sucrose by GS and GSGB in the absence of dex-
tran (Fig. 3A,B). For this purpose, we incubated
sucrose (50 mm) with GS or GSGB (0.4 lm) in the
absence of dextran, and analyzed the products at arbi-
trary time intervals. The time courses of the produc-
tion of insoluble glucan and other sugars by GSGB
were essentially the same as those for GS (Fig. 3).
Fig. 1. Schematic structures of GTF-I and its variant proteins. The
proteins used start at Asp85 of GTF-I and terminate at Ser1085 and
Asn1592 for GS and GSGB, respectively. Both proteins contain an
extra peptide, TMITNSSSVPG, from the multiple cloning site of
pUC18 at their N-terminal ends. The black bars in the GBd indicate
the ‘A’ repeat [6,7].
Kinetics ofStreptococcussobrinus GTF-I reaction H. Komatsu et al.
532 FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS
In the initial phase ( 60 min), the concentration of
fructose increased (open squares in Fig. 3) and the
concentration of released glucose was lower than that
of fructose (open circles in Fig. 3). As a result, the
difference between the concentrations of fructose and
glucose increased with time, suggesting that glucosyl
transfer occurred. Leucrose, a known GTF-I product
[25,26], was detected after 30 min, and its concentra-
tion increased over time (open triangles in Fig. 3).
Finally, the concentration of insoluble glucan increased
with a lag time of 60 min (filled squares in Fig. 3). The
glucosidic linkages of the insoluble glucan produced by
GS or GSGB were analyzed with
13
C-NMR spectros-
copy (Fig. S1). The spectra confirm that both of the
glucans were a-(1fi 3)-linked glucose polymers. The
release of the sugars flattened off after 100 min, indi-
cating sucrose depletion.
After all of the sucrose (50 mm) had been consumed,
35 mm fructose, 12 mm glucose, 12 mm leucrose and
12 mm (glucose equivalent) insoluble glucan were pro-
duced. The total concentrations of fructose and glu-
cose were approximately 50 mm (resulting from 35 mm
fructose and 12 mm leucrose) and 35–40 mm (resulting
from 12 mm glucose, 12 mm leucrose, and 12 mm
insoluble glucan), respectively. The fructose yield was
equivalent to the initial amount of sucrose, whereas
the glucose yield was significantly less. The 10–15 mm
glucose that went undetected appears to have been
incorporated into soluble nigerooligosaccharides.
A
B
Fig. 2. Effect of dextran on the synthesisof insoluble glucan and
glucosyl transfer activity by GS and GSGB. (A) Dextran-dependent
insoluble glucan synthesis. The insoluble glucan synthesis reaction
was initiated by adding GSGB (filled squares) or GS (open squares)
to the sucrose solution in the presence of dextran. The amount of
insoluble glucan was monitored by light scattering. The reaction
mixture contained 50 n
M GTFs, 50 lgÆmL
)1
dextran T2000 and
100 m
M sucrose in 10 mM Mops (pH 7.0). (B) Glucosyl transfer
activity as a function of dextran concentration. The reaction mixture
contained 10 n
M GTFs, 50 mM sucrose, 100 mM NaCl, and 10 mM
Mops (pH 6.8). Glucosyl transfer velocity was estimated as
described in Experimental procedures. Filled and open circles indi-
cate the GTF activity of GSGB and GS, respectively.
A
B
Fig. 3. Saccharide production kinetics during the dextran-indepen-
dent GTF-I reaction. (A) and (B) show the production of saccharide
by GS and GSGB, respectively. Open squares, open circles, open
triangles and filled squares indicate the production of fructose,
glucose, leucrose, and insoluble glucan, respectively. The produc-
tion of insoluble glucan is plotted as the glucose equivalent molar
concentration. The starting solution contained 0.4 l
M enzyme (GS
or GSGB) and 50 m
M sucrose in 10 mM potassium phosphate
(pH 6.8).
H. Komatsu et al. KineticsofStreptococcussobrinus GTF-I reaction
FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS 533
To detect the soluble nigerooligosaccharides, soluble
products were analyzed with HPLC, but no peak cor-
responding to nigerooligosaccharides was detected
(data not shown). In addition, TLC detected major
spots of fructose, glucose, and leucrose; immobile spots
and diffuse smears corresponding to a degree of poly-
merization (DP) ‡ 4 were observed after 50 min
(Fig. S2). These results suggest that the nigerooligo-
saccharides were marginally accumulated and that they
constituted a small proportion of the oligosaccharides
with a wide range of DP values.
Glucosyl transfer activity of GS and GSGB for
nigerooligosaccharide acceptors
Next, we estimated the glucosyl transfer activity of
acceptor reactions for methyl a-d-glucopyranoside,
nigerooligosaccharides (DP = 2–6), and leucrose
(Fig. 4). Methyl a-d-glucopyranoside was used as a
glucose analog that is not a substrate of hexokinase in a
glucose assay system. The data were analyzed on the
basis of the Michaelis–Menten equation; the maximum
activity (k
cat
) and Michaelis constant for acceptor
(K
m
Acc
) values are listed in Table 1. Methyl a-d-glucopyr-
anoside and nigerooligosaccharides served as the gluco-
syl acceptors for GS and GSGB; in contrast, leucrose
was not a glucosyl acceptor. The activity for
nigerooligosaccharides was higher than that for methyl
a-d-glucopyranoside. Furthermore, the nigerooligo-
saccharides with higher DPs exhibited higher activity.
The rank order of glucosyl transfer efficiencies (k
cat
⁄
K
m
Acc
) was as follows: nigerohexaose > nigeropen-
taose = nigerotetraose > nigerotriose = nigerose >
methyl a- d-glucopyranoside. In addition, the kinetics of
the acceptor reactions appeared to be similar between
GS and GSGB.
Kinetic simulation of GTF glucan synthesis
To explain the time course of saccharide production
during the synthesisof insoluble glucan, we assumed
Scheme 1, which shows the intermediate products that
are the probable acceptors for the GTF-I reaction in
the absence of dextran. GTF-I was assumed to be capa-
ble of catalyzing the following reactions: (a) the hydro-
lysis of sucrose to glucose and fructose (reaction 1);
(b) the transfer of glucose residues from sucrose to
glucose and nigerooligosaccharide to produce higher-
DP nigerooligosaccharide (reactions 2–7); and (c) the
transfer of glucose residues from sucrose to fructose to
produce leucrose (reaction 8). Here, we assumed that
nigerooligosaccharides with DP ‡ 7 were insoluble,
because the solubility of nigeroheptaose (DP = 7)
was extraordinarily low (submillimolar level) at neu-
tral pH.
For numerical simulation, we made two assump-
tions: (a) Michaelis–Menten kinetics for sucrose
hydrolysis, as described above and indicated by Kraij
et al. [21] for reuteransucrase; and (b) ping-pong bi-bi
A
B
Fig. 4. GTF activity as a function of acceptor concentrations. The
initial velocity of glucosyl transfer by GS (A) and GSGB (B) was
measured in the presence of leucrose (open diamonds), methyl
a-
D-glucopyranoside (filled circles), nigerose (open circles), nigerotri-
ose (filled squares), nigerotetraose (open squares), nigeropentaose
(filled triangles), and nigerohexaose (open triangles). The lines indi-
cate the curve fitted to the Michaelis–Menten equation to obtain
k
cat
and K
m
Acc
(Table 1), with the nonlinear regression program in
the
ORIGIN software package (v. 6.1J). The data from the GS nigero-
pentaose reaction were analyzed by nonlinear least-squares analy-
sis, but the parameters were not obtained with reasonable
accuracy. Because the solubility of nigerooligosaccharides (DP ‡ 4)
was very low, GTF activity could not be measured at higher con-
centrations.
Kinetics ofStreptococcussobrinus GTF-I reaction H. Komatsu et al.
534 FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS
kinetics for glucosyl transfer from sucrose to an
acceptor sugar, as indicated for other glucansucrases
[5,18,19] and fructansucrase [22], and confirmed for
the nigerotriose acceptor reaction by GS (Fig. S3).
With these assumptions, the glucan synthesis process
was numerically simulated on the basis of Scheme 1,
with the experimentally estimated kinetic parameters
(Table 2). The characteristic time course obtained
from the experimental data was reproduced by this
simulation (Fig. 5): (a) the fructose concentration
increased at an appreciably higher rate than the glu-
cose concentration, until all of the sucrose was con-
sumed – the difference between the concentrations of
fructose and glucose subsequently increased with time;
(b) insoluble glucan (DP ‡ 7) production increased
after a lag phase ( 50 min); (c) small quantities of
each nigerooligosaccharide (DP = 2–6) were pro-
duced (Fig. 5B); (d) leucrose production increased
with a delay time ( 30 min); and (e) the final yield
of saccharide was similar to that of the experimental
data, even in terms of the total concentration of
soluble nigerooligosaccharides (DP = 2–6). In other
words, the difference between the total concentrations
of fructose and glucose (50 mm and 35–40 mm,
respectively) can be explained by this simulation.
The simulation also indicates that the difference is
attributable to the production of 10–15 mm soluble
nigerooligosaccharides.
Table 1. Kinetic parameters of the acceptor reaction. The parameters were obtained by nonlinear least-squares curve-fitting analysis for the
data presented in Fig. 4. The k
cat
and K
m
Acc
were estimated from the maximum activity and the Michaelis constant for the acceptor, respec-
tively. ND, not determined.
Acceptor
GS GSGB
k
cat
(s
)1
)
K
m
Acc
(mM)
k
cat
⁄ K
m
Acc
(s
)1
ÆmM
)1
)
k
cat
(s
)1
)
K
m
Acc
(mM)
k
cat
⁄ K
m
Acc
(s
)1
ÆmM
)1
)
Methyl a-
D-glucopyranoside 39 ± 8
a
70 ± 31 0.56 ± 0.27 41 ± 25 78 ± 65 0.53 ± 0.54
Nigerose 53 ± 5 27 ± 5 2.0 ± 0.4 41 ± 4 12 ± 3 3.4 ± 0.9
Nigerotriose 57 ± 3 26 ± 3 2.2 ± 0.3 69 ± 9 33 ± 10 2.1 ± 0.7
Nigerotetraose 73 ± 8 16 ± 4 4.6 ± 1.2 65 ± 12 17 ± 8 3.8 ± 1.9
Nigeropentaose ND ND 59 ± 19 13 ± 9 4.5 ± 3.5
Nigerohexaose 53 ± 3 5 ± 1 11 ± 2 40 ± 3 6 ± 1 6.7 ± 1.2
a
Errors of the estimates are indicated.
Scheme 1. Kinetic model ofdextran-independent insoluble glucan synthesis.
H. Komatsu et al. KineticsofStreptococcussobrinus GTF-I reaction
FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS 535
Discussion
No significant difference was observed with respect to
dextran-independent insoluble glucan synthesis and the
nigerooligosaccharide acceptor reaction between GS
and GSGB (Figs 3 and 4); therefore, the dextran-
independent synthesisofa-(1fi3)-glucan is not an
artificial reaction catalyzed by the deficient protein,
but rather a basal reaction of GTF-I. In contrast, in the
presence of dextran, the enzymatic activity of GSGB
was much higher than that of GS (Fig. 2). Dextran-
dependent glucan synthesis probably takes place via
enhancement of the basal reaction of the GSd by the
binding of dextran to the GBd. This result is consistent
with the idea that the GBd plays an important role in
dextran-dependent glucan synthesis [2,11–13].
The experimental time course of saccharide produc-
tion agreed well with the simulation based on
Scheme 1, using experimentally determined kinetic
parameters. This agreement, in turn, supports the
appropriateness of the model in Scheme 1; thus, our
results provide a reasonable explanation for the dex-
tran-independent glucan synthesis reaction. At the
beginning of the reaction, sucrose is hydrolyzed to glu-
cose and fructose. The released glucose subsequently
serves as the glucosyl acceptor to form nigerose. The
subsequent glucosyl transfers take place to nigerooligo-
saccharides to form higher-DP nigerooligosaccharides.
In this manner, insoluble glucan is formed by the elon-
gation of nigerooligosaccharides.
Efficient glucan synthesis, such that the accumulation
of intermediate nigerooligosaccharides (DP = 2)6) is
minimized, is explainable by the single-chain mecha-
nism, whereby the enzyme ‘continuously’ elongates only
one acceptor molecule until the release of the resulting
glucan [15,16]. However, because the K
m
Acc
values were
relatively high (5–80 mm; Table 1) as compared with the
concentrations of acceptors (glucose and nigerooligo-
saccharides) produced under the experimental condi-
tions, the enzyme tends to release the nigerooligo-
saccharide during the reaction. This mode is consistent
with nonprocessive elongation, whereby the enzyme
releases the products after each transfer of a glucose
residue to the acceptor. Hence, efficient insoluble glucan
synthesis with minimal accumulation of intermediate
nigerooligosaccharides is not achieved by continuous
(or processive) elongation; it is, rather, achieved mainly
through the enzymatic kinetic properties of GTF-I, the
catalytic efficiency of which increases with an increase in
the DP of nigerooligosaccharide (Table 1). In fact, we
demonstrated that the enzyme kinetic model can simu-
late the time course of insoluble glucan synthesis. Non-
processive elongation was previously demonstrated for
the formation of insoluble amylase-like polymers from
sucrose by Neisseria polysaccharea amylosucrase [23].
Table 2. Kinetic parameters for the numerical simulation. Simula-
tion parameters were estimated as described in Doc. S1.
Reaction
k
cat
(s
)1
)
K
m
Suc
(mM)
K
m
Acc
(mM)
k
(s
)1
ÆmM
)1
)
Sucrose hydrolysis 10 0.3
Acceptor reaction
Glucose 40 2 70
Nigerose 50 2 20
Nigerotriose 60 2 30
Nigerotetraose 70 2 20
Nigeropentaose 60 2 10
Nigerohexaose 50 2 6
Fructose 0.02
A
B
Fig. 5. Kinetic simulation ofdextran-independent glucan synthesis
by GTF-I. The time course of saccharide production was simulated
on the basis of the enzyme kinetic parameters listed in Table 2.
(A) Time course of sucrose and major products. Molar concentra-
tions of sucrose (filled circles), fructose (open squares), glucose
(open circles) and leucrose (open triangles) are indicated. The con-
centrations of soluble nigerooligosaccharides (open diamonds) and
insoluble glucan (filled squares) are converted into glucose equivalent
molar concentrations. Nigerooligosaccharides with DP ‡ 7 were
defined as insoluble glucan. (B) Nigerooligosaccharide production.
The amounts of nigerose (circles), nigerotriose (squares), nigerotetra-
ose (diamonds), nigeropentaose (triangles) and nigerohexaose
(inverted triangles) are indicated as molar concentrations.
Kinetics ofStreptococcussobrinus GTF-I reaction H. Komatsu et al.
536 FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS
The improved catalysis in the presence of increasing DP
may be a common feature of glucansucrase.
Additional studies are required to elucidate the dex-
tran-dependent synthesisof glucan. As the affinity of
the GBd of GTF-I for dextran is extraordinarily high
(K
m
= 4.88 · 10
7
m
)1
) [27], the enzyme can hardly be
dissociated from dextran. GTF-I may elongate the
a-(1 fi 3)-glucose branches of dextran in a processive-
like manner. Moulis et al. propose a semiprocessive
mechanism of polymerization for Leuconostoc mesen-
teroides NRRL B-512F dextransucrase and L. mesen-
teroides NRRL B-1355 alternansucrase [24]. According
to this mechanism, the glucan-binding domains at the
C-terminal end of GTFs act as mediators of the shift
between the processive and nonprocessive processes;
GTF-I may otherwise randomly migrate on the dextran
without dissociating during the reaction. Recently, high-
speed atomic force microscopy demonstrated that
Trichoderma reesei cellobiohydrolase I moves along
crystalline cellulose in a processive manner [28]; there-
fore, to understand the mechanism underlying the
dextran-dependent GTF-I reaction, the dynamics of
binding of GTF-I to dextran should be investigated with
the use of single-molecule measurements, surface plas-
mon resonance, and ⁄ or quartz crystal microbalances.
This study defines, for the first time, the relationship
between the kinetic properties of GTF and the time
course of saccharide production. Owing to GTF’s
kinetic property of improving activity in the presence
of nigerooligosaccharides with increasing DPs, inter-
mediate nigerooligosaccharides minimally accumulate
during production, and insoluble glucan is efficiently
produced. This feature may be favorable for dental
plaque formation by mutans streptococci, because the
efflux of the intermediate product may be diminished
in an oral environment. Moreover, such kinetic analy-
sis could also provide useful information regarding the
GTF-I-catalyzed synthesisofa-(1fi 3)-linked glucose
polymers for industrial production of foods, pharma-
ceuticals, and cosmetics [4,29].
Experimental procedures
Plasmids
Plasmid pGS [14] was used for the expression of GS
(Asp85–Ser1085). To construct the GSGB (Asp85–
Asn1592)-encoding plasmid pGSGB6R, an EcoRI–BglII
fragment (1.5 kbp) encoding a part of the multiple cloning
site and Asp85–Glu596 was isolated from pAB2 [6] and
inserted into a BglII–EcoRI-digested pAB1 [6] fragment
(3.4 kbp) encompassing the Asp597–Asn1592 coding
region. Both plasmids contained pUC18-derived DNA
(containing the lac promoter and the ampicillin resistance
gene) and encoded an extra peptide, TMITNSSSVPG, from
the multiple cloning site at the N-terminal end.
Enzymes
GS was prepared from Escherichia coli JM109 transformed
with pGS. Protein expression and purification were carried
out as described previously [14]. GSGB was expressed in
E. coli JM109 transformed with pGSGB6R, and prepared
with the same procedure as that used for GS. GSGB was
further purified by gel filtration chromatography on a
Toyopearl HW-55 column (TOSOH) (2.5 · 90 cm) pre-
equilibrated in 0.5 m NaCl and 10 mm potassium phos-
phate (pH 6.8). The protein concentration was determined
from the absorbance at 280 nm, with a molar extinction
coefficient calculated from the amino acid composition [30].
Preparation ofa-(1fi 3)-glucan
GS was added to 300 mm sucrose solution containing
0.02% NaN
3
and 10 mm potassium phosphate (pH 6.8),
giving a final concentration of 100 nm. The mixture was
kept at room temperature until no more reducing sugars
were produced (approximately 1 week). The resulting insol-
uble material was collected by decanting and centrifugation
at 600 g for 4 min, and was subsequently washed with
water by suction filtration. The structure of the product
was confirmed by
13
C-NMR spectroscopy.
Preparation of nigerooligosaccharides
The a-(1fi3)-glucan described above was subjected to mild
acid hydrolysis (0.05 m H
2
SO
4
at 80 °C for 2 h) to release the
small amount of fructose ( 1%) contained in the glucan.
The fructose-free glucan was then subjected to limited acid
hydrolysis (0.1 m H
2
SO
4
at 100 °C for 100 min) to produce
nigerooligosaccharides. After the hydrolysis was stopped by
neutralization with NaHCO
3
, the insoluble material was
removed by centrifugation at 500 g for 4 min. Finally, the
soluble fraction was applied to a column (4 · 22 cm) con-
taining equivalent weights of activated carbon (Wako Pure
Chemical, Osaka, Japan) and Celite (Wako Pure Chemical)
(Fig. S4). The column was maintained at 15 °C, and was
then washed with water until the glucose was completely
eluted. The bound oligosaccharides were eluted with a gradi-
ent of 0–8% butanol. The sugars were detected by use of the
phenol ⁄ H
2
SO
4
method [31], and characterized by TLC.
Measurement of glucosyl transfer velocity
The glucosyl transfer rates were measured as described
by Konishi et al. [14]. The initial velocities of all GTF
reactions were measured for the first 4–8 min at 25 °C. The
H. Komatsu et al. KineticsofStreptococcussobrinus GTF-I reaction
FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS 537
reaction mixtures contained dextran, nigerooligosaccha-
rides, methyl a-d-glucopyranoside and leucrose at various
concentrations in 10 nm enzyme, 50 m m sucrose, 100 mm
NaCl, and 10 mm Mops (pH 6.8). After the concentrations
of the resultant glucose and fructose were measured, the
extent of sucrose splitting was determined from the amount
of fructose released, and the extent of glucosyl transfer was
calculated by subtracting the amount of free glucose from
the amount of free fructose.
For kinetic analysis, the assay was carried out with at
least four different concentrations of each acceptor [methyl
a-d-glucopyranoside and nigerooligosaccharide (DP = 2–6)].
The kinetic parameters (velocity constant, k; Michaelis
constant, K
m
) were determined with the nonlinear regres-
sion program in the origin software package (v. 6.1J)
(OriginLab, Northampton, MA, USA).
Light scattering
The formation of insoluble glucan was monitored by light
scattering at 90° in a thermostated cell (25 °C) at a wave-
length of 350 nm. The reaction mixture contained 50 nm
GTFs, 50 lgÆmL
)1
dextran T2000 and 100 mm sucrose in
10 mm Mops (pH 7.0).
Analysis of products during the glucan synthesis
reaction
GS or GSGB (0.4 lm) and 50 mm sucrose were incubated
in 10 mm potassium phosphate (pH 6.8) at 25 °C. A suit-
able volume of mixture was sampled at various time points,
and the reaction was stopped by addition of NaOH at a
final concentration of 10 mm. After the samples had been
immediately centrifuged at 18,000 g for 10 min, the insolu-
ble glucan was recovered as a precipitate and washed with
water. The supernatants were neutralized with HCl. The
precipitates were completely hydrolyzed in 1.2 m HCl at
approximately 100 °C for 50 min, and this was followed by
neutralization with 1.2 m NaHCO
3
. The hydrolysate was
then subjected to the dinitrosalicylic acid method [32] to esti-
mate the amount of insoluble glucan in terms of glucose. For
the analysis of soluble sugars in the supernatants, the follow-
ing methods were employed: (a) glucose and fructose concen-
trations were determined with the enzymatic assay [33]; and
(b) leucrose and nigerooligosaccharide were detected by
TLC, and their concentrations were determined by HPLC.
Simulation of the glucan synthesis reaction
The synthesisof insoluble glucan was numerically analyzed
on the basis of Scheme 1. We assumed Michaelis–Menten
kinetics for sucrose hydrolysis [14,21], and ping-pong
bi-bi kinetics for the glucosyl transfer from sucrose to an
acceptor sugar [5,18,19,22]. Using the quasi-steady-state
assumption, we obtained a set of 10 differential equations
that describe the time course of saccharide production. The
fourth-order Runge)Kutta method with a 3.6-min step was
used to solve the 10 differential equations, with the kinetic
parameters estimated from the experimental data using
visual basic for applications in Microsoft Excel (http://
chemeng.on.coocan.jp). All parameters used are listed in
Table 2. The details of these differential equations and the
simulation parameter are described in Doc. S1.
13
C-NMR analysis
To inhibit base-catalyzed reactions, the insoluble glucan
was dissolved at 60 mgÆ mL
)1
in 0.5 m NaOD in D
2
O con-
taining 3.5 mgÆmL
)1
NaBD
4
. The
13
C-NMR analyses were
performed on a JEOL JNM-A500 spectrometer (Center for
Instrumental Analysis, Kyushu Institute of Technology).
Spectra were recorded at 125.65 MHz at room temperature,
with an acquisition time of 0.9667 s and 8000 scan accumu-
lations. Chemical shifts are expressed in parts per million,
with 3-trimethylsilyl-1-propanesulfonic acid sodium salt as
a reference. Peaks were assigned according to the method
outlined in Colson et al. [34].
HPLC
Leucrose and nigerooligosaccharide concentrations were
measured by HPLC with a YMC-pack polyamine II column
(4.6 · 250 mm) (YMC, Kyoto, Japan). The column was
maintained at 30 °C, and the flow rate was 0.5 mLÆmin
)1
.
The eluents were water ⁄ acetonitrile (25 : 75, v ⁄ v) and
water ⁄ acetonitrile (30 : 70, v ⁄ v) for the analysis of disaccha-
rides (leucrose and nigerose) and nigerooligosaccharides
(DP = 3–5), respectively. Carbohydrates were detected with
a differential refractometer. Concentrations were calculated
from the peak area of the refractive index, using a calibration
curve. The sample for leucrose analysis was incubated with
yeast invertase (0.2 mgÆmL
)1
)at35°C for 30 min to con-
sume sucrose before injection, because leucrose was eluted at
7.7 min, which was relatively close to sucrose (7.4 min).
TLC
After the samples were concentrated on a centrifugal evap-
orator, they were separated by TLC, with three ascents on
Silica Gel 70 plates (Wako Pure Chemical) in 15 : 3 : 4
(v ⁄ v ⁄ v) 1-butanol ⁄ pyridine ⁄ water [35]. The sugars were
detected by spraying the plates with H
2
SO
4
, and then heat-
ing at 120 °C for 5 min.
Acknowledgements
We thank H. Sakamoto (Kyushu Institute of Technol-
ogy) for providing access to the apparatus for
Kinetics ofStreptococcussobrinus GTF-I reaction H. Komatsu et al.
538 FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS
biochemical experiments. We appreciate the assistance
provided by M. Ikeguchi, Y. Fujita and T. Kawauchi
for nigerooligosaccharide preparation and separation.
This work was supported in part by grants-in-aid for
Scientific Research (B) 13557161 and (C) 13671904 (to
K. Fukui), (C) 13680744 (to T. Kodama) and a grant-
in-aid for Young Scientists (B) 13080537 (to H. Koma-
tsu) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan. The initial parts of
this work were performed at the Institute for Materials
Chemistry and Engineering, Kyushu University.
References
1 Loesche WJ (1986) Role ofStreptococcus mutans in
human dental decay. Microbiol Rev 50, 353–380.
2 Monchois V, Willemot RM & Monsan P (1999) Glu-
cansucrase: mechanism of action and structure–function
relationships. FEMS Microbiol Rev 23, 131–151.
3 Simpson CL, Giffard PM & Jacques NA (1995) Strep-
tococcus salivarius ATCC 25975 possesses at least two
genes coding for primer-independent glucosyltrans-
ferases. Infect Immun 63, 609–621.
4 van Hijum SA, Kralj S, Ozimek LK, Dijkhuizen L &
van Geel-Schutten IG (2006) Structure–function rela-
tionships of glucansucrase and fructansucrase enzymes
from lactic acid bacteria. Microbiol Mol Biol Rev 70,
157–176.
5 Mooser G (1992) Glycosidases and glycosyltransferases.
Enzymes 20, 187–231.
6 Abo H, Matsumura T, Kodama T, Ohta H, Fukui K,
Kato K & Kagawa H (1991) Peptide sequences for
sucrose splitting and glucan binding within Streptococ-
cus sobrinusglucosyltransferase (water-insoluble glucan
synthetase). J Bacteriol 173, 989–996.
7 Ferretti JJ, Gilpin ML & Russell RR (1987) Nucleotide
sequence of a glucosyltransferase gene from Strepto-
coccus sobrinus MFe28. J Bacteriol 169, 4271–4278.
8 Wong C, Hefta SA, Paxton RJ, Shively JE & Mooser
G (1990) Size and subdomain architecture of the
glucan-binding domain of sucrose:3-alpha-D-glucosyl-
transferase from Streptococcus sobrinus. Infect Immun
58, 2165–2170.
9 Fukui K, Moriyama T, Miyake Y, Mizutani K &
Tanaka O (1982) Purification and properties of
glucosyltransferase responsible for water-insoluble
glucan synthesis from Streptococcus mutans. Infect Im-
mun 37, 1–9.
10 Koga T, Sato S, Inoue M, Takeuchi K, Furuta T &
Hamada S (1983) Role of primers in glucan synthesis
by glucosyltransferases from Streptococcus mutans strain
OMZ176. J Gen Microbiol 129, 751–754.
11 Kato C, Nakano Y, Lis M & Kuramitsu HK (1990)
Carboxyl-terminal deletion analysis of the Streptococcus
mutans glucosyltransferase-I enzyme. FEMS Microbiol
Lett 72, 290–302.
12 Monchois V, Arguello-Morales M & Russell RRB
(1999) Isolation of an active catalytic core of
Streptococcus downei MFe28 GTF-I glucosyltransferase.
J Bacteriol 181, 2290–2292.
13 Kingston KB, Allen DM & Jacques NA (2002) Role of
the C-terminal YG repeats of the primer-dependent
streptococcal glucosyltransferase, GtfJ, in binding to
dextran and mutan. Microbiology 148, 549–558.
14 Konishi N, Torii Y, Yamamoto T, Miyagi A, Ohta H,
Fukui K, Hanamoto S, Matsuno H, Komatsu H, Kod-
ama T et al. (1999) Structure and enzymatic properties
of genetically truncated forms of the water-insoluble
glucan-synthesizing glucosyltransferase from Streptococ-
cus sobrinus. J Biochem (Tokyo)
126, 287–295.
15 Robyt JF, Kimble BK & Walseth TF (1974) The mech-
anism of dextransucrase action. Direction of dextran
biosynthesis. Arch Biochem Biophys 165, 634–640.
16 Robyt JF, Yoon SH & Mukerjea R (2008) Dextransucr-
ase and the mechanism for dextran biosynthesis. Carbo-
hydr Res 343, 3039–3048.
17 Mooser G, Hefta SA, Paxton RJ, Shively JE & Lee TD
(1991) Isolation and sequence of an active-site peptide
containing a catalytic aspartic acid from two Strepto-
coccus sobrinus alpha-glucosyltransferases. J Biol Chem
266, 8916–8922.
18 Kobayashi M & Matsuda K (1978) Inhibition of
dextran synthesisby glucoamylase and endodextranase.
Carbohydr Res 66, 277–288.
19 Mooser G, Shur D, Lyou M & Watanabe C (1985)
Kinetic studies on dextransucrase from the cariogenic
oral bacterium Streptococcus mutans. J Biol Chem 260,
6907–6915.
20 Mukasa H, Shimamura A & Tsumori H (2000) Nige-
rooligosaccharide acceptor reaction of Streptococcus
sobrinus glucosyltransferase GTF-I. Carbohydr Res 326,
98–103.
21 Kralj S, van Geel-Schutten GH, van der Maarel MJ &
Dijkhuizen L (2004) Biochemical and molecular charac-
terization of Lactobacillus reuteri 121 reuteransucrase.
Microbiology 150, 2099–2112.
22 Chambert R, Treboul G & Dedonder R (1974) Kinetic
studies of levansucrase of Bacillus subtilis. Eur J
Biochem 41, 285–300.
23 Albenne C, Skov LK, Mirza O, Gajhede M, Feller G,
D’Amico S, Andre
´
G, Potocki-Ve
´
rone
`
se G, van der
Veen BA, Monsan P et al. (2004) Molecular basis of
the amylose-like polymer formation catalyzed by
Neisseria polysaccharea amylosucrase. J Biol Chem 279,
726–734.
24 Moulis C, Joucla G, Harrison D, Fabre E, Potocki-
Veronese G, Monsan P & Remaud-Simeon M (2006)
Understanding the polymerization mechanism of
H. Komatsu et al. KineticsofStreptococcussobrinus GTF-I reaction
FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS 539
glycoside-hydrolase family 70 glucansucrases. J Biol
Chem 281, 31254–31267.
25 Monchois V, Vignon M, Escalier PC, Svensson B &
Russell RR (2000) Involvement of Gln937 of Strepto-
coccus downei GTF-I glucansucrase in transition-state
stabilization. Eur J Biochem 267, 4127–4136.
26 Monchois V, Vignon M & Russell RR (2000) Mutagen-
esis of asp-569 ofglucosyltransferaseI glucansucrase
modulates glucan and oligosaccharide synthesis. Appl
Environ Microbiol 66, 1923–1927.
27 Komatsu H, Katayama M, Sawada M, Hirata Y, Mori
M, Inoue T, Fukui K, Fukada H & Kodama T (2007)
Thermodynamics of the binding of the C-terminal
repeat domain ofStreptococcussobrinus glucosyltrans-
ferase-I to dextran. Biochemistry 46, 8436–8444.
28 Igarashi K, Koivula A, Wada M, Kimura S, Penttila
¨
M
& Samejima M (2009) High speed atomic force micros-
copy visualizes processive movement of Trichoderma
reesei cellobiohydrolase I on crystalline cellulose. J Biol
Chem 284, 36186–36190.
29 Hellmuth H, Wittrock S, Kralj S, Dijkhuizen L, Hofer
B & Seibel J (2008) Engineering the glucansucrase
GTFR enzyme reaction and glycosidic bond specificity:
toward tailor-made polymer and oligosaccharide prod-
ucts. Biochemistry 47, 6678–6684.
30 Gill SC & von Hippel PH (1982) Calculation of protein
extinction coefficients from amino acid sequence data.
Anal Biochem 182, 319–326.
31 Dubois M, Gilles KA, Hamilton JK, Robers P &
Smith F (1956) Colorimetric method for determination
of sugars and related substances. Anal Chem 28, 350–
356.
32 Chaplin MF (1986) Monosaccharides. In Carbohydrate
Analysis: Practical Approach (Chaplin MF & Kennedy
JF eds), pp 1–36. IRL Press, Oxford.
33 Schmidt FH (1961) Die enzymatische Bestimmung von
Glucose und Fructose nebeneinander. Klin Wochenschr
39, 1244–1247.
34 Colson P, Jarrell HC, Lamberts BL & Smith IC (1979)
Determination, by carbon-13 nuclear magnetic reso-
nance spectroscopy, of the composition of glucans syn-
thesized by enzymes of the cariogenic organism
Streptococcus mutans. Carbohydr Res 71, 265–272.
35 Mukasa H, Tsumori H & Shimamura A (2001) Dextran
acceptor reaction ofStreptococcussobrinus glucosyl-
transferase GTF-I as revealed by using uniformly
13
C-labeled sucrose. Carbohydr Res 333, 19–26.
Supporting information
The following supplementary material is available:
Fig. S1.
13
C-NMR analysis of the insoluble glucan
produced by GS and GSGB.
Fig. S2. TLC analysis of saccharide production by GS
and GSGB.
Fig. S3. Double reciprocal plot of nigerotriose accep-
tor reaction of GS.
Fig. S4. Separation of nigerooligosaccharides by acti-
vated carbon chromatography.
Doc. S1. Simulation of the glucan synthesis reaction.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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should be addressed to the authors.
Kinetics ofStreptococcussobrinus GTF-I reaction H. Komatsu et al.
540 FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS
. Kinetics of dextran-independent a-(1
fi
3)-glucan synthesis
by Streptococcus sobrinus glucosyltransferase I
Hideyuki Komatsu
1
, Yoshie Abe
1
, Kazuyuki. Owing to GTF’s
kinetic property of improving activity in the presence
of nigerooligosaccharides with increasing DPs, inter-
mediate nigerooligosaccharides