Kinetics of dextran-independent a-(1
fi
3)-glucan synthesis
by Streptococcus sobrinus glucosyltransferase 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

doi:10.1111/j.1742-4658.2010.07973.x
Glucosyltransferase (GTF)-I from cariogenic Streptococcus sobrinus elon-
gates the a-(1 fi 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-(1 fi 3)-glucan from sucrose in the
absence of the pre-existing dextran (i.e. in a dextran-

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 Streptococcus sobrinus 6715 (serotype g)
(Fig. 1). A numerical simulation based on the enzyme
kinetic parameters is in good agreement with the time
course of dextran-independent insoluble glucan forma-
tion. These results suggest that the dextran-independent
synthesis of insoluble glucan by GTF-I proceeds via
the nonprocessive elongation of a-(1 fi 3)-linked glu-
cose polymers (nigerooligosaccharides).
Results
Functional characterization of GSGB
We previously investigated the functional role of the
GBd in dextran-dependent a-(1 fi 3)-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-(1 fi 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

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 of Streptococcus sobrinus 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-

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. Kinetics of Streptococcus sobrinus 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

) 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 synthesis of 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

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

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 of dextran-independent insoluble glucan synthesis.
H. Komatsu et al. Kinetics of Streptococcus sobrinus 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 synthesis of a-(1 fi 3)-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

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)

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 of Streptococcus sobrinus 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 synthesis of 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

(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 of a-(1 fi 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

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. Kinetics of Streptococcus sobrinus 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

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-

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 of Streptococcus sobrinus 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 of Streptococcus 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.

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

Veronese G, Monsan P & Remaud-Simeon M (2006)
Understanding the polymerization mechanism of
H. Komatsu et al. Kinetics of Streptococcus sobrinus 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 of glucosyltransferase I 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 of Streptococcus sobrinus 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-

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
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
Kinetics of Streptococcus sobrinus GTF-I reaction H. Komatsu et al.
540 FEBS Journal 278 (2011) 531–540 ª 2010 The Authors Journal compilation ª 2010 FEBS