Download Glucansucrases: mechanism of action and structure–function

FEMS Microbiology Reviews 23 (1999) 131^151
Glucansucrases: mechanism of action and structure^function
relationships
Vincent Monchois, Reneè-Marc Willemot, Pierre Monsan *
Centre de Bioingeènierie Gilbert Durand, UMR CNRS 5504, LA INRA, INSA, Complexe Scienti¢que de Rangueil,
31077 Toulouse Cedex 4, France
Received 21 April 1998; received in revised form 12 October 1998 ; accepted 19 November 1998
Abstract
Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic
acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named
glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight
oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not
necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological
importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase
encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood.
However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation
of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of
glucansucrase encoding genes in addition to structure^function relationship studies have allowed the identification of
important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca.
1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a
series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action
mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure^function
relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional
structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which
determine function. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.
Keywords : Dextransucrase; Glucosyltransferase ; Leuconostoc mesenteroides ; Streptococcus; Glucan binding; Glycosylhydrolase;
Oligosaccharide
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Mechanism of glucansucrase action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +33 (5) 6155-9415; Fax: +33 (5) 6155-9399; E-mail: [email protected]
0168-6445 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 4 1 - 2
FEMSRE 642 16-4-99
132
134
132
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
2.1. Glucan biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1. Initiation of the reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2. Elongation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.1. Elongation occurring at the non-reducing end of the glucan chain
2.1.2.2. Elongation occurring at the reducing end of the glucan chain . . .
2.2. Acceptor reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Oligosaccharide synthesis reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1.1. Type of acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1.2. Mechanism of acceptor reaction . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Glucan as an acceptor: mechanism proposed for branching . . . . . . . . . . .
3. Structural and functional organisation of the glucansucrases . . . . . . . . . . . . . . . . . . .
3.1. The N-terminal end of glucansucrases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The presence of two functional domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Structure^function relationships of the N-terminal catalytic domain . . . . . . . . . . . . .
4.1. Identi¢cation of the catalytic site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Identi¢cation of other important regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Secondary structure prediction of the N-terminal catalytic domain . . . . . . . . . . .
5. Structure^function relationships of the C-terminal glucan binding domain . . . . . . . . .
5.1. Structure of the C-terminal domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Role of the C-terminal domain in glucan binding . . . . . . . . . . . . . . . . . . . . . . .
5.3. Role of the C-terminal domain in glucansucrase activity . . . . . . . . . . . . . . . . . .
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References
...............................................................
1. Introduction
Glucansucrases (EC 2.4.5.1) are extracellular enzymes mainly produced by the soil bacterium Leuconostoc mesenteroides (commonly termed dextransucrase), Streptococcus species from the oral £ora
(commonly termed glucosyltransferases) and by lactic bacteria Lactococci [1]. They catalyse the synthesis of high molecular weight D-glucose polymers
named glucans from sucrose. When e¤cient acceptors, like maltose or isomaltose, are added to the reaction medium, glucansucrase catalyses the synthesis
of low molecular weight oligosaccharides instead of
high molecular weight glucan [2].
Di¡erent kinds of glucans with di¡erent sizes and
structures, depending on the glucansucrase-producing strain, are synthesised [1,3,4]. They present a
main linear chain composed of D-glucopyranosyl
units, either principally linked through K(1^6) glucosidic bonds (dextran polymer), or through K(1^3)
glucosidic bonds (mutan polymer) as well as linked
alternately through K(1^6) and K(1^3) glucosidic
bonds (alternan polymer). Glucans also di¡er in
the type of branch linkages, resulting from K(1^2),
K(1^3), K(1^4) and K(1^6) glucosidic bonds, the de-
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
134
135
135
135
135
137
137
137
137
138
138
138
139
139
139
140
141
145
145
145
146
146
147
gree of branching, the length of branch chains and
their spatial arrangement.
Dextransucrases from L. mesenteroides are used in
industry. Dextran produced by L. mesenteroides
NRRL B-512F was one of the ¢rst biopolymers to
be produced on an industrial scale in 1948 [5] and to
¢nd several applications in medicine, separation
technology and biotechnology [6,7]. Oligosaccharides
produced by L. mesenteroides NRRL B-1299 with
one or more D-glucopyranosyl units linked through
K(1^2) glucosidic bonds [8] are highly resistant to
attack by digestive enzymes [9] and are used as prebiotics in cosmetic and human nutritional applications, as they are speci¢cally metabolised by bene¢cial saprophite £ora.
Glucansucrases produced by oral streptococci, like
Streptococcus mutans and Steptococcus sobrinus, play
a key role in the cariogenesis process, as the glucan
produced enhances the attachment and colonisation
of cariogenic bacteria [10,11]. In order to develop
vaccines against dental caries, studies for the isolation of the genes coding for these enzymes were initiated more than 10 years ago. The sequences of 14
di¡erent glucansucrase encoding genes (named gtf)
are now available [12^33] (Table 1).
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
133
Table 1
Main characteristics of glucansucrases from Streptococcus mutans and L. mesenteroides
Strain
Gene
S. mutans GS5
gtf-B
gtf-C
gtf-D
S. mutans LM7
gtf-C
S. downei Mfe28
gtf-I
gtf-S
Glucana
87%
13%
85%
15%
30%
70%
K(1^3)
K(1^6)
K(1^3)
K(1^6)
K(1^3)
K(1^6)
nd
88%
12%
10%
90%
K(1^3)
K(1^6)
K(1^3)
K(1^6)
Size (aa)
Mr b (103 )
Primerc
References
1475
150
3
[12,13]
1375
140
3
[14,15]
1430
155
+
[16,17]
1375
150
+
[18]
1556
160
+
[19^21]
1328
147
3
[19,22]
S. sobrinus 6715 (serotype g)
gtf-Ia
nd
1592
160
+
[23]
S. sobrinus OMZ176 (serotype d)
gtf-T
27% K(1^3)
73% K(1^6)
nd
1542
163
3
[24,25]
1590
175
+
[26]
90%
10%
100%
50%
50%
5%
95%
K(1^3)
K(1^6)
K(1^6)
K(1^3)
K(1^6)
K(1^3)
K(1^6)
1522
168
+
[27^29]
1599
1490
176
157
+/3
3
[27^30]
[29,31,32]
1576
171
3
[29,32]
gtf-Is
S. salivarius ATCC 25975
gtf-J
gtf-K
gtf-L
gtf-M
S. gordonii (S. sanguis)
gtf-G
40% K(1^3)
60% K(1^6)
1578
170
3
[33]
L. mesenteroides NRRL B-512F
dsr-S
5% K(1^3)
95% K(1^6)
1527
170
3
[34,84]
L. mesenteroides NRRL B-1299
dsr-A
15% K(1^3)
85% K(1^6)
5%K(1^3)
95% K(1^6)
1290
146
3
[35]
1508
167
3
[36]
dsr-B
Glucan characterisations were achieved using glucansucrases produced by expression of genes cloned in E. coli.
Structure of the produced glucan. nd, not determined. Percentages are percentages of total linkages in glucan structure.
b
Molecular weight deduced from protein sequences.
c
Activator e¡ect (+) or not (3) of an exogenous glucan.
a
Sequence information for gtf and for the more
recently cloned L. mesenteroides glucansucrase encoding genes (dsr) [34^36] show that they are closely
related and have a common structure. They code for
large enzymes with an average molecular weight of
160 000. These proteins present an N-terminal
conserved catalytic domain of about 900 aa and a
C-terminal domain covering 300^400 aa composed
of a series of homologous, directly repeating units
and responsible for glucan binding. Some studies
have allowed the precise identi¢cation of the regions
essential for enzyme catalysis. However, key elements de¢ning the regioselectivity and stereospeci¢city of glucansucrases remain to a large part unknown
FEMSRE 642 16-4-99
134
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
Fig. 1. Reactions catalysed by glucansucrases. I, Glucan synthesis by successive transfer of glucosyl units ; II, sucrose hydrolysis by transfer of the glucosyl unit onto water ; III, oligosaccharide synthesis by transfer of the glucosyl unit onto an acceptor molecule ; and IV, isotopic exchange by reverse reaction of glucosyl^enzyme complex formation.
and, up to now, no crystallographic data are available for glucansucrases.
This review is focused on the di¡erent mechanisms
proposed for the di¡erent reactions catalysed by glucansucrases, as well as on the di¡erent data provided
by structure^function relationship studies carried out
on these enzymes by using molecular biology techniques.
2. Mechanism of glucansucrase action
Glucansucrases catalyse transfer of glucosyl residues coming from sucrose cleavage. The synthesis of
di¡erent products by glucansucrases depends on the
destination of these glucosyl units [37] (Fig. 1). The
dextran synthesis reaction occurs by successive transfer of glucosyl units to the polymer. In the presence
of acceptor molecules in the reaction medium, the
transfer of the glucosyl units is made onto these
molecules, leading to oligosaccharide synthesis (acceptor reaction). Glucansucrases can also transfer
glucosyl units onto water molecules and simply hydrolyse sucrose.
Whatever the glucosyl units' destination is, their
transfer supposes ¢rst the formation of a covalent
glucosyl^enzyme complex [38^40] which has been
isolated by Mooser and Iwaoka [41] for the glucansucrase produced by S. sobrinus. Except for the formation of the covalent glucosyl^enzyme complex, the
reverse reaction being called isotopic exchange, the
other reactions are mainly irreversible and are in
competition with the reactive intermediary glucosyl^enzyme [42].
Sucrose is the only natural donor substrate for this
synthesis [43]. Sucrose might induce a change in protein conformation that might activate the catalytic
site [37]. Energy necessary to catalyse all the reactions comes only from the cleavage of the glucosidic
bond of sucrose. The mediation of nucleotide-activated sugars or cofactors is not necessary. The reaction occurs with a retention of the K-con¢guration of
the anomer during the cleavage of the glucosidic
bond [44].
2.1. Glucan biosynthesis
Glucan synthesis occurs by autopolymerisation
and is a single chain mechanism: this elongation is
catalysed by a unique type of enzyme. No oligosaccharide is detected at the beginning of the reaction.
Isolated glucan at a low rate of sucrose conversion,
already presented a high molecular weight [45,46].
Tsuchiya et al. [46] have proposed a mechanism in
which glucan chains were tightly bound with the enzyme during their elongation. It has been proposed
that glucan is bound to the enzyme through noncovalent links [38]. More recently, results coming
from inhibition kinetic studies using various agents
suggested that the glucan binding site was separated
from the sucrose binding site [47^51]. This hypothesis has also been con¢rmed by the identi¢cation of
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
glucansucrase sequences and by structure^function
relationship studies (see Section 3).
Tsuchiya et al. [46] proposed that the glucan synthesis mechanism may be separated in three di¡erent
steps: (1) initiation; (2) elongation; and (3) termination. The last step corresponds to the dissociation of
the glucan from the enzyme.
2.1.1. Initiation of the reaction
The necessity of a primer to start the polymerisation has been often discussed. The necessity of a
primer was ¢rst accepted in relation to glycogen synthesis mechanism studies carried out at the same
period [43]. This hypothesis has been supported by
the fact that addition of exogenous glucan has an
activating e¡ect on glucan synthesis [50,52,53]. However, glucansucrases are active enzymes in the absence of any exogenous primer. Robyt and Corrigan
[54] have shown that a dextran presenting blocked
hydroxyl groups linked to C6 of the glucosyl residue
located at its non-reducing end remained a strong
activator of glucan synthesis. Exogenous glucan
seems not to play the same role of primer as glycogen or starch for amylophosphorylases. Its activator
e¡ect may be due to a conformational e¡ect [55].
Binding of glucan to enzyme may promote a change
in the catalytic site conformation allowing the enzyme to become more active [37].
2.1.2. Elongation step
The mechanism of autopolymerisation and direction of chain growth remains an aspect still not fully
understood.
2.1.2.1. Elongation occurring at the non-reducing
end of the glucan chain
An action mechanism similar to that of glycosidases has been proposed for glucansucrases (Fig.
2A) [37,50,56]. This mechanism involved the presence of an aspartic (or glutamic) acid acting as a
nucleophilic group and another residue acting as a
proton donor [57]. The carboxyl group might make a
nucleophilic attack on the C1 of the glucosyl moiety
of sucrose, leading to the formation of a covalent
glucosyl^enzyme complex. The other acidic group
might facilitate the release of fructose by giving a
proton to the oxygen atom involved in the glucosidic
link. It also enables another glucosyl residue to be
135
activated by trapping the hydrogen from the hydroxyl group linked to the C6 .
If glucan biosynthesis follows this proposed mechanism, the result is that elongation occurs at the nonreducing end of the glucan chain. Only one covalent
glucosyl^enzyme complex is then required. This
mechanism has been considered as irrelevant by Su
and Robyt [58] because a presence of a primer initiating the glucan synthesis reaction is required in
this mechanism and, according to them, sucrose
seems not to be able to participate in the chain initiation.
2.1.2.2. Elongation occurring at the reducing end of
the glucan chain
Ebert and Schenk [38] were the ¢rst to propose a
mechanism where glucan elongation occurred at its
reducing end. The glucan chain may grow by successive insertions of glucose units between the enzyme
catalytic site and the reducing end of the polysaccharide.
Robyt et al. [39] have shown by pulse-chase experiments using sucrose labelled with 14 C that glucan
and glucosyl residues coming from sucrose cleavage
were both covalently linked with enzyme through
their reducing end. Similar experiments carried out
with S. mutans 6715 glucansucrase [59] or with an
immobilised glucansucrase free of glucan from S.
sanguis ATCC 10558 [60] led to the same conclusion:
chain growth occurs at the reducing end.
To explain how glucan elongation occurs at the
reducing end, Robyt et al. [39] have proposed a
mechanism in which they suggested that two identical nucleophilic sites are involved (Fig. 2B). This
mechanism is composed of two distinct steps: (1)
the two nucleophilic sites attack two sucrose molecules leading to the release of two fructose molecules
and giving two glucosyl residues covalently linked to
the enzyme. The mechanism of the formation of
these two covalent glucosyl^enzyme complexes may
be identical to the action mechanism of glycosidases;
and (2) the OH-C6 of one of the two glucosyl residues may make a nucleophilic attack on the C1 of
the other one. That promotes the formation of an
K(1^6) bond and the release of one of the two nucleophilic sites which can attack another sucrose in order to create a new covalent glucosyl^enzyme complex.
FEMSRE 642 16-4-99
136
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
Fig. 2. Mechanisms of action proposed for glucansucrases. (A) Mechanism involving one nucleophilic site. (B) Mechanism involving two
nucleophilic sites. X, nucleophilic group; A, proton donor group.
Glucan elongation occurs by the repetition of this
mechanism. The two catalytic sites are alternately
involved in covalent complexes with glucosyl residue
or glucan (Fig. 2B). At each step, a glucosyl residue
is inserted at the reducing end of the glucanosyl^
enzyme complex. Fructose may act as an acceptor
to the glucanosyl chain linked to the enzyme, result-
ing in the chain termination [39]. However, up to
now, the presence of fructose at the reducing end
of glucan has never been reported.
An interesting aspect of this mechanism is that it
allows a glucan elongation by its reducing end without requiring the presence of exogenous primer at
the beginning of the reaction. A kinetic study
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
achieved by Germaine and Schachtele [61] with glucansucrase from S. mutans 6715 concluded that two
sucrose binding sites, but only one in the presence of
glucan, existed in this enzyme. Another study, in
which glucansucrase from S. sanguis ATCC 10558
was photolabelled using p-azidophenyl-K-D-glucopyranoside (APG), has suggested that there were
two substrate binding sites [62]. E¡ect of the binding
of 6-deoxysucrose, a strong inhibitor of glucansucrase, with L. mesenteroides NRRL B-512F enzyme
tended also to show that two sucrose binding sites
really existed [58].
However, up to now, only one site (being an aspartic acid) capable of making a covalent bond with
the glucose moiety coming from the breakdown of
sucrose has been clearly identi¢ed [41]. Considering
the sequential insertion, it is also di¤cult to understand how the OH-C6 of one glucosyl group is able
to attack the C1 of the dextransosyl group when it is
far away (Fig. 2B), except if a con¢guration inversion occurs.
So despite intensive work carried out for more
than 25 years to elucidate the mechanism of glucansucrase action, none of the proposed mechanisms
appears to be the relevant one.
2.2. Acceptor reaction
The acceptor reaction was ¢rst described by Koepsell et al. [2]. In the presence of sucrose, they have
observed that introduction into the reaction medium
of molecules, like maltose, isomaltose, and O-Kmethylglucoside, shifted the pathway of glucan synthesis towards the production of oligosaccharides.
Moreover, glucan itself can act as an acceptor molecule to yield a branched polysaccharide.
2.2.1. Oligosaccharide synthesis reaction
2.2.1.1. Type of acceptors
The acceptor reaction has been studied with dextransucrases from L. mesenteroides and more particularly L. mesenteroides NRRL B-512F. Although
dextransucrases accept a limited number of donor
substrates other than sucrose [63^65], numerous sugars can act as acceptors [2,66]. A speci¢city of recognition seems to exist. Yamamauchi and Ohawada
[67] showed that K,K-trehalose, but neither K,L-trehalose nor L,L-trehalose, acted as acceptors. More-
137
over, galactose, the limit dextrins produced by glucoamylase, or amino acid residues and peptides are
not acceptors [2].
These di¡erent acceptors may be shared into two
main classes, according to their ability to compete
with glucan synthesis or their e¡ect on the reaction
velocity: (1) the strong acceptors, like maltose or
isomaltose, have an activator e¡ect on the reaction
velocity and strongly inhibit the yield of glucan synthesis [66]; and (2) the weak acceptors, like fructose
or melibiose, have an inhibitory e¡ect on the reaction [2]. The yield of oligosaccharides produced is
low as well [66].
Some acceptors, for instance disaccharides such as
maltose or oligosaccharides presenting an isomaltosyl residue at their non-reducing end, allow a series
of oligosaccharides to be produced [68]. Synthesis
progresses by successive transfers of glucosyl units
to oligosaccharides which are alternately product
and substrate. Other acceptors, like fructose [69], allow only one oligosaccharide (leucrose) to be produced in which the glucosyl residue coming from
the breakdown of sucrose is added to the acceptor.
The leucrose synthesis reaction, in fact, becomes important at the end of the glucan synthesis reaction
when the fructose concentration is high.
2.2.1.2. Mechanism of acceptor reaction
Robyt and Walseth [68] proposed that only one
covalent glucosyl^enzyme complex was necessary
and that acceptor molecules were incorporated at
the reducing end of the glucan or the oligosaccharide
produced. Oligosaccharide elongation might occur at
the reducing end. According to the mechanism proposed by Robyt et al. [39] for glucan synthesis, oligosaccharides may be synthetised by a nucleophilic
attack of the hydroxyl group located at the non-reducing end of the acceptor to the C1 of one of the
two glucosyl residues involved in the two covalent
glucosyl^enzyme complexes.
Various experiments tended to show that the acceptor binding site is really unique and separated
from the two active sites [58,70], but, up to now,
there is no direct evidence that a separate acceptor
binding exists. Moreover, according to Germaine
and Schachtele [61] and Kobayashi and Matsuda
[71], one of the two sucrose binding sites may also
be an acceptor binding site. In DSR-S from L. mesenteroides NRRL-B 512F, change of Asp-551, being
FEMSRE 642 16-4-99
138
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
the site of glucosyl^enzyme complex formation, in
asparagine, resulted in a total loss of both glucan
and oligosaccharide synthesis activities [84].
The activator e¡ect of maltose on the reaction
velocity may be explained by a change in the limiting
step occurring during the acceptor reaction. In the
presence of maltose, the formation of the glucosyl^
enzyme complex becomes the limiting step instead of
the polymer transfer in the case of the glucan synthesis reaction [72]. Mayer et al. [73] attributed this
activator e¡ect to a change in the enzyme conformation resulting of acceptor binding with dextransucrase. The reason why weak acceptors, such as fructose, inhibit the reaction remains mostly unclear. An
earlier hypothesis supported by Koepsell et al. [2]
was that these acceptors inhibited sucrose breakdown. More recently, Boker et al. [74] proposed
that the presence of fructose may create a steric hindrance inhibiting the growth of the glucan chain.
2.2.2. Glucan as an acceptor: mechanism proposed for
branching
Glucan can also be regarded as an acceptor.
Mayer et al. [73] noticed an increase in the molecular
weight of exogenous glucan by acceptor reaction.
The ¢nal structure of the glucan produced by S.
mutans 6715 necessitated the action of several glucansucrases [75]. The transfer of glucose and glucan
to a glucan chain acceptor have been noticed with
the dextransucrase produced by L. mesenteroides
NRRL B-512F [76]. Also, the phenomenon of insolubilisation of exogenous glucan by formation of
K(1^3) linkages has also been noticed with di¡erent
glucansucrases [73,77^80].
As suggested by Ebert and Brosche [81] in early
work, K(1^3) branch point formation may be the
result of acceptor reaction. Robyt and Taniguchi
[76] reported that an K(1^3) linkage was created between the anomeric carbon involved in one covalent
glucosyl^enzyme complex and the OH-C3 of a glucosyl residue of the exogenous glucan. They proposed that exogenous glucan may bind in another
site and one of the hydroxyl groups may exert a
nucleophilic attack on the C1 involved in the glucosyl^ or glucanosyl^enzyme complex. However, this
mechanism, based upon an acceptor reaction mechanism, remains insu¤cient to explain the synthesis of
highly branched glucan [82]. Cote and Robyt [83]
suggested that K(1^3) linkages of a highly branched
glucan may be synthesised by transfer of glucosyl
residues from a glucosyl^enzyme site di¡erent from
the two active sites to the glucan.
3. Structural and functional organisation of the
glucansucrases
Studies initiated more than 10 years ago allowed
the isolation and sequencing of 17 genes coding for
glucansucrases produced by oral streptococci involved in the cariogenesis process, S. mutans GS5
or LM7, S. sobrinus serotype h (S. downei Mfe28),
serotype g (S. sobrinus 6715), serotype d (S. sobrinus
OMZ176), by S. salivarius ATCC 25975 and by L.
mesenteroides NRRL B-512F and B-1299 (Table 1)
[12^36]. Isolated glucansucrases are all large enzymes
with an average Mr of 160 000. Streptococci glucansucrases synthesise primarily K(1^3) rich mutan polysaccharides and K(1^6) rich dextran polysaccharides
[12^33]. L. mesenteroides glucansucrases produce
K(1^6) rich dextrans [34^36,84]. However, genes coding for glucansucrases-producing branched glucan
through K(1^2) or K(1^4) glucosidic bond or synthesising alternan have not yet been isolated.
Analysis and comparison of the di¡erent protein
sequences show that these enzymes are closely related and have a common structure (Fig. 3). They
are composed of four distinct structural domains
[30,32,33,85]. Except for DSR-A from L. mesenteroides NRRL B-1299 [35], their N-terminal end begins with a signal peptide of 32^34 aa followed by a
stretch of 123^129 amino acids which is highly variable. They present a highly conserved core region of
about 1000 amino acids. This domain, also named
catalytic domain, is the sucrose binding domain, capable of binding and cleaving sucrose. The C-terminal end of glucansucrases covering about 500 amino
acids is composed of a series of tandem repeats and
constitutes the glucan binding domain.
3.1. The N-terminal end of glucansucrases
Glucansucrase signal peptides are typical signal
peptides of Gram-positive bacteria. They consist of
a basic N-terminal part followed by a hydrophobic
core region and a more polar C-terminal region [86].
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
139
Fig. 3. Schematic structure of glucansucrases for which encoding genes have been cloned. A, signal peptide ; B, variable region; C, N-terminal catalytic domain; D, C-terminal glucan binding domain.
The hydrophobic region is one of the rare hydrophobic regions present in glucansucrases, these proteins being overall hydrophilic in nature [13,15,17].
The main characteristic of the structure of glucansucrase signal peptides is that it is well conserved, sharing a 42% identity [36]. A lower identity level also
exists with the signal peptide of a fructosyltransferase secreted by S. mutans GS-5 [87], although there is
no similarity between these two groups of enzymes
[17]. This shared identity between signal peptides
may indicate that these enzymes use the same secretion pathway in oral streptococci and Leuconostoc
mesenteroides.
The non-conserved region located just downstream of the signal peptide seems to have no important role in the enzyme mechanism. Its deletion does
not a¡ect the enzyme activity [23]. Moreover, DSRA, isolated from L. mesenteroides NRRL B-1299 is
an active enzyme that does not possess this variable
region [35]. The signi¢cance of this variable region
also remains unknown.
3.2. The presence of two functional domains
Ferretti et al. [21] ¢rst demonstrated that the glucan binding domain was distinct from the domain
involved in sucrose splitting and that it was located
at the C-terminal end. Cloning of the 3P-end of gtf-I
from S. downei Mfe28 in frame fusion with lacZ
allowed non-active 65-kDa protein to be expressed
able to bind to Sepharose 1000. Truncation of the
C-terminal end conducted to suppress glucan binding abilities of GTF-I [21]. Mooser and Wong [88] as
well as Kobayashi et al. [89] have isolated by mild
trypsic digestion of S. sobrinus GTF-S and GTF-I,
peptides of 60.5 or 55 kDa, respectively, having the
same a¤nity for glucan as native enzyme, but displaying no activity. Sequencing of peptides from
GTF-I of S. sobrinus showed that it corresponded
to the C-terminal part. Moreover, Abo et al. [23]
have isolated a non-active 60-kDa peptide able to
bind glucan and corresponding to the C-terminal
end of GTF-Ia. So, the C-terminal part covering
about the last 500 aa is a functional domain able
to bind glucan itself.
Ferretti et al. [21] suggested that the N-terminal
domain was the catalytic domain. However, C-terminal truncations led to express glucansucrases unable
to produce polymer [21,23,90,91]. In some cases, a
sucrose cleavage activity is retained [90]. The necessary presence of at least a part of the C-terminal
domain to have a glucansucrase fully active also
has been reported for a cellobiohydrolase produced
by Trichoderma reesei presenting a similar two domain (catalytic and binding with cellulose) structure
[92].
4. Structure^function relationships of the N-terminal
catalytic domain
As shown by protein sequence alignments of different glucansucrases, the N-terminal domain is
highly conserved (Fig. 4). However, these sequence
alignments have not allowed the identi¢cation of
consensus sequences associated with a particular
type of activity (e¡ect of primer addition, type of
linkage produced, among others) [30,32]. This shows
that the knowledge of glucansucrase primary sequences is not su¤ciently developed for investigating structure^function relationships. Moreover, due
perhaps to the large size of these enzymes, no
crystallographic data are available and only few
structure prediction studies have been carried out
[93,94].
4.1. Identi¢cation of the catalytic site
The group of Mooser has demonstrated that a site
making a covalent link with the glucosyl residue
FEMSRE 642 16-4-99
140
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
coming from sucrose breakdown is present inside the
N-terminal domain. Mooser and Iwakoa [41] have
isolated from S. sobrinus GTF-S a covalent glucosyl^enzyme complex. Mooser et al. [95] have isolated
a peptide from GTF-I and GTF-S of S. sobrinus
presenting the active site. The corresponding sequences were very similar and contained three aspartic
acids. Mass spectrometry analysis revealed that the
Asp residue inside the peptide coming from GTF-I
(Asp^Ser^Ile^Arg^Val^Asp--Ala^Val^Asp) made an
ester bond with the glucosyl residue. This peptide
was located at 449 aa from the N-terminal end of
GTF-I. Site directed mutagenesis experiments have
con¢rmed the importance of this amino acid (Fig.
4). Replacement of homologous Asp residues in the
GTF-B sequence (Asp-451) by Thr, Asn and Gln [96]
and in the GTF-I sequence from S. downei Mfe28
(Asp-453) by Asn [94] completely suppressed the enzyme activity. Change of Asp-551 in Asn for DSR-S
also abolished the activities of glucan and oligosaccharide synthesis [84]. The mutations do not a¡ect
glucan binding ability of these two enzymes, GTF-B
and DSR-S [84,96]. Other mutations a¡ecting Asp
residues near Asp-451 from GTF-B had no signi¢cant e¡ect on enzymatic activity [96]. The functional
importance of this region has also been con¢rmed by
the fact that an antibody directed against a peptide
of sequence homologous to this region inhibited glucansucrase activity [97^100].
With respect to an action mechanism similar to
the glycosidase action mechanism, Mooser et al.
[95] suggested that at least one more amino acid
has to be involved in the catalytic process to facilitate the fructose release by playing the role of proton
donor.
The involvement of histidines in the enzymatic
mechanism has been illustrated by photooxidation
experiments where only histidine residues were modi¢ed [101] or by using diethylpyrocarbonate (DEP)
[102,103]. Fu and Robyt [102] also concluded from
inhibition curves with DEP that two histidines were
involved in the action mechanism of glucansucrases.
Site-directed mutagenesis experiments have enabled
functionally important conserved His to be located
(Fig. 4). The replacement of His-661 of DSR-S [84]
and its equivalent in GTF-B (His-561) [104] to Arg
led to a very weakly active enzyme.
4.2. Identi¢cation of other important regions
The region preceding the active site seems to be
essential for the biological activity. A genetic polymorphism among S. mutans serotype c strains a¡ecting the region extending from aa 387 to 427 in the
GTF-B sequence or from aa 413 to 453 in the GTFC sequence does not induce a change in the amino
acid sequence [105,106] (Fig. 4). An antibody directed against a peptide whose sequence was homologous to the 435-453 GTF-C region has an inhibitory e¡ect on GTF-C and GTF-B activity [107].
However, this inhibitory e¡ect is not observed with
GTF-D where this peptide sequence is also present.
Chia et al. [107] suggested that the antibody may
inhibit branching or that signi¢cant conformational
di¡erences may exist for this region in GTF-B or
GTF-C and GTF-D.
Funane et al. [103] reported the existence of a
second active site for the dextransucrase produced
by L. mesenteroides NRRL B-512F in a region
with a sequence homologous to the 435^453 GTFC region (corresponding to the 509^527 DSR-S region). They showed that the modi¢cation of carboxylic groups with 1-ethyl-3-(3-dimethyl-amino)propylcarbdodiimide (EDC) and glycine ethyl ester (GEE),
this latter compound playing the role of nucleophile,
abolished glucansucrase activity. Since addition of
sucrose delayed the enzyme inactivation, authors
have concluded that these modi¢ed carboxyl groups
may be involved in the binding with the substrate. In
order to identify them, a ¢rst EDC-GEE inactivation
was carried out in the presence of sucrose monocaprate followed by a second inactivation using EDC
and a £uorescent nucleophilic agent ((N-1-naphtyl)ethylenediamine, EDAN) instead of GEE. A £uorescent peptide was isolated after mild trypsic digestion
of the modi¢ed enzyme. Its was determined as sequence, LQEDNSNVVVEA, and shares 58% identity with a highly conserved region in glucansucrase
enzymes, corresponding to the 436^447 GTF-C region and contains one Asp and two Glu residues
which were suggested as providing an essential carboxyl group [103]. While it is apparent that the peptide sequence di¡ers from the highly conserved sequence identi¢ed in all glucansucrases by nucleotide
sequencing, the only region of GTF sequence with
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
which this aligns is the conserved region corresponding to the 436^447 GTF-C region. It seems likely
that the di¡erence is due to technical problems associated with peptide sequencing. The presence of functional carboxyl groups in this region has been con¢rmed by site-directed mutagenesis experiments (Fig.
4). Substitution of Asp-511 and Asp-513 of DSR-S
in Asn resulted, respectively, in a complete suppression and a strong decrease in the glucan and oligosaccharide synthesis activities [84]. The substitution
of the similar amino acids (Asp-411 and Asp-413) of
GTF-B in Asn showed that Asp-413 was essential,
but not Asp-411, a still active enzyme being obtained
after mutation [104]. The two GTF-B variants exhibited Km values similar to the wild-type GTF-B, suggesting that the two Asp residues were not directly
involved in the binding of sucrose [104].
In addition, Dertzbaugh and Macrina [108] have
shown that an upstream region, the 342^356 GTF-B
region was also important (Fig. 4). An antibody directed against a peptide homologous to this region
inhibits the synthesis of both soluble and insoluble
glucan. Inhibition studies of dextransucrase produced by L. mesenteroides NRRL B-512F with
o-phthaldehyde (OPA) also suggested that lysines
are essential for activity [109,110]. One of these essential lysines may be close of the active site [111].
Replacement of four conserved Tyr residues at position 169^172 in GTF-B suggested that this region
(Fig. 4), presenting a signi¢cant homology with repeating units thought to be involved in glucan-binding, may play a role in the enzymatic mechanism
[104].
Site-directed mutagenesis experiments have also
allowed the identi¢cation of amino acid residues in£uencing the structure of the glucan produced. By
comparing GTF-I, GTF-S, GTF-B, GTF-C and
GTF-D sequences, Shimamura et al. [112] have identi¢ed positions where amino acid residues are conserved for the enzymes producing an insoluble glucan but di¡erent from the residues present in the
enzymes producing a soluble glucan. Ile-448, Asp457, Asp-567, Lys-779 and Lys-1014 present in
GTF-B, an enzyme-producing insoluble glucan,
have been chosen to be mutated in the corresponding
amino acid present in GTF-D, an enzyme producing
a soluble glucan: Val-462, Asn-471, Thr-589, Gln810 and Thr-1046, respectively [112] (Fig. 4). Only
141
the mutation D567T enabled a change in the structure of the glucan produced by GTF-B to be
achieved: the percentage of K(1^3) linkages is 38%,
instead of 76% with the wild-type enzyme. The
mutation, T589D in GTF-D, has also an e¡ect on
the glucan structure, the mutated enzyme producing
an insoluble one [112]. However, even though this
study clearly shows the direct involvement of these
residues in the glucan structure determination, the
mechanism de¢ning their involvement remains to
be elucidated.
4.3. Secondary structure prediction of the N-terminal
catalytic domain
Ferretti et al. [21] have suggested that GTF-I possesses sequences homologous with K-amylases. The
degree of homology is low, but approximates to that
with typical sequences of enzymes belonging to the
K-amylase superfamily [93]. The N-terminal domain
secondary structure prediction studies carried out by
MacGregor et al. [93] and Devulapalle et al. [94]
tend to show that glucansucrases possess a (K/L)8
barrel structure, like glycosidases (including K-amylase), cyclodextrin glucanotransferase (CGTase), isoamylase, and a glucan glucosidase of S. mutans [113].
This motif, ¢rst identi¢ed for triose phosphate isomerase is found in many proteins having various
functions and displaying little or no homology
[114]. This motif is characterised by the presence of
8 L-strands (E1^E8) located in the core of the protein alternated with 8 K-helices (H1^H8) located at
the surface of the protein.
These two structure prediction studies have given
a similar result concerning the location of H3^E8
[93,94] (Fig. 4). The aspartic acid involved in the
covalent glucosyl^enzyme complex may be located
near the C-terminal end of E4. However, H1^E3
location is not identical for these two structure predictions. For MacGregor et al. [93], a circular permutation of these elements occurred in glucansucrases: the NH2 terminal helix may be the H3
element and the next elements, E1^H1^E2^H2^E3,
may be far away in the sequence instead of being
located at the beginning (Fig. 4). According to Devulapalle et al. [94], these ¢rst elements may be located in the N-terminal variable region of glucansucrases. However, this last hypothesis is not consistent
FEMSRE 642 16-4-99
142
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
FEMSRE 642 16-4-99
143
Fig. 4. For legend please see p. 114.
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
FEMSRE 642 16-4-99
144
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
Fig. 4. Sequence alignment of the N-terminal conserved domain of glucansucrases. Alignment was made using ClustalW. Sequences shown
are these from GTF-B [13], GTF-C [15], GTF-D [17], GTF-I [21], GTF-S [22], GTF-T [25], GTF-J [28], GTF-K [30], GTF-M [32], GTFG [33], DSR-S [34], DSR-A [35] and DSR-B [36]. About 10% of the residues are identical. , identical or conserved residues in all sequences in the alignment; :, conserved substitutions; W, semi-conserved substitutions ; - - -, gap in the sequence; aa-aa-aa-aa-aa, sequence of
peptides used to generate antibodies [107,108]; aa, amino acid having been mutated; t, putative catalytic residues [93,94]; F, putative residues stabilising the transition state [93]; b, putative calcium binding sites [94]; W, putative chloride binding sites [94]; E, putative residues which may play a role in the binding with acceptor molecules and in the transfer of the glucosyl residue. [93]; -Ex- and -Hx-, localisation of L-strands (E) and K-helices (H) according to MacGregor et al. [93]. Dotted horizontal lines indicate that there is a sequence
gap between two adjacent blocks.
6
with the fact that DSR-A is active and does not
possess this region [35].
These structure predictions allow the alignment of
conserved amino acid residues in GTFs with amino
acid residues playing a role in action mechanism of
glycosylases. It seems that Glu-475, Asp-547 and
Asp-437 of GTF-S may be involved in the catalytic
mechanism [93,94] (Fig. 4). Substitution of these residues in GTF-I supported this prediction, since it led
to the inhibition of enzyme activity [94]. His-546 and
Gln-920 of GTF-S may stabilise the transition step.
Asp-440, Asn-441, Ala-476, Trp-477 and Ser-478 of
GTF-S may play a role in the binding with acceptor
molecules and in the transfer of the glucosyl residue
[93]. The substitution of homologous Trp residue in
GTF-B (Trp-491) resulted in an enzyme devoid of
activity and supported the hypothesis that this residue may play a role in the action mechanism of
GTFs [104]. The role of calcium binding for Asp397 and of chloride binding for Arg-435 of GTF-S
have been assigned [94].
Table 2
Comparison of the pattern of repeated units composing the C-terminal domain of glucansucrases
Strain
Gene
Repeating units
References
S. mutans GS5
gtf-B
gtf-C
gtf-D
A-A-C-A-C-A-C-A-C-A-C-A-C
A-A-C-A-C-A-C-A
A-A-A-A-A
[13]
[15]
[17]
S. downei Mfe28
gtf-I
gtf-S
A-A-C-A-C-A-C-B-A-C-B-A-C
A-A-C-A-C-A-C
[21]
[22]
S. sobrinus 6715 (serotype g)
gtf-Ia
A-A-C-A-C-A-C-A-C-A-C
[23]
S. sobrinus OMZ176 (serotype d)
gtf-T
gtf-Is
A-A-C-A-C-C-A-A-C
A-A-C-A-C-A-C-A-C-A-C
[25]
[26]
S. salivarius ATCC 25975
gtf-J
gtf-K
gtf-L
gtf-M
A-D-A-D-A-D
A-D-A-A-A-D-A-D-A-D-A-D
A-A-C-A-C-A-C
A-C-A-C-A-A-C-A
[28]
[30]
[32]
[32]
S. gordonii (S. sanguis)
gtf-G
A-A-C-A-C-A-C-A-C-A-C
[33]
L. mesenteroides NRRL B-512F
dsr-S
A-C-C-A-A-C
[34,115]
L. mesenteroides NRRL B-1299
dsr-A
dsr-B
A-A-A-C-A-C-A-C
A-C-C-A-A-C
[35]
[36]
Consensus sequences [21,28,85] are the following for:A repeated units, WYYFNXDGQAATGLQTIDGQTVFDDNGXQVG ; B repeated
units, VNGKTYYFGSDGTAQTQANPKGQTFKDGSVLRFYNLEGQYVSGSGWY ; C repeated units, GKIFFDPDSGEVVKNRFV;
and D repeated units, GGVKNADGTYSKY.
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
5. Structure^function relationships of the C-terminal
glucan binding domain
5.1. Structure of the C-terminal domain
Sequence analysis of di¡erent glucansucrases
showed that these enzymes all presented a C-terminal domain composed of a series of repeated units.
They have been divided into four major classes: A,
B, C and D repeats [21^23,28]. The number and
distribution of these repeats is speci¢c to each enzyme (Table 2). However, it appears that D repeated
units are speci¢c to enzymes produced by S. salivarius ATCC 25975. A units are always present, often in
an A^C pattern [85]. The C-terminal ends of DSR-S
and DSR-B are characterised by the presence of
small repeated units, not very homologous to each
other [34,36,115]. The exact involvement of these different patterns in glucan structure determination has
not been elucidated [33]. However, except for GTFL, enzymes producing an insoluble glucan possess
the same pattern, A-A-C-A-C-A-C (Table 2).
Similar repeated units composed of A^C motif are
present in a glucan binding protein produced by S.
mutans Ingbritt [116]. They can also be found in the
C-terminal part of a dextranase inhibitor protein
produced by S. sobrinus UAB 108 acting like a glucan binding protein [117,118]. C-Terminal domains
of Clostridium di¤cile toxins A and B as well as
some lytic enzymes from S. pneumoniae present similar repeated units [119,120]. The C-terminal domain
of toxin A is involved in the binding with an oligosaccharide (GalK-3GalL-4GlcNac), component of
the receptors of target cells [121].
Wren [119] showed that A repeats found in the
C-glucan binding domain of glucansucrases and in
C. di¤cile toxins always present three conserved amino acid residues: a tyrosine^phenylalanine dipeptide
and a glycine located ten residues downstream. Sequence comparison enabled a consensus sequence for
these repeated units to be established. According to
von Eichel-Streiber et al. [122], they are composed of
the same amino acid residue pattern consisting of an
aromatic amino acid residue stretch, sometimes including a tyrosine, surrounded by conserved residues. The following consensus sequence has been
proposed [122]: IDGYYFD+N+G. More recently,
another consensus motif named YG repeat has
145
been presented [123]: NDGYYFxxxGxxHO x(G/
N)xHO HO HO . (x, non-conserved amino acid residue;
HO , hydrophobic amino acid residue). It is found
both within the A^D repeats and also outside
them. It includes an aromatic amino acid stretch
surrounded by neutral or polar amino acid residues,
such as a conserved glycine residue. Its end is composed of hydrophobic residues [123].
5.2. Role of the C-terminal domain in glucan binding
The C-terminal domain is responsible for the binding of glucan. Conserved amino acid residues may be
involved in binding with glucosidic units. The clustered aromatic residues (tyrosine, tryptophane and
phenylalanine) may stabilise the binding between
sugar and protein by interacting with the sugar
unit [124]. The polar (lysine, glycine and phenylanine) or acid (aspartic acid) residues may allow
the creation of hydrogen bonds with hydroxyl residues of the sugar [124]. The presence of amino acid
residues, like lysine, glycine, asparagine or serine,
able to introduce £exibility into the protein structure,
may allow the glucosyl residue to be correctly orientated to the binding sites [125].
Singh et al. [126] showed by using di¡erent chemical inhibitors, that tyrosine, tryptophan, histidine
and aspartic (or glutamic) acid residues were important for the binding abilities of glucansucrases. After
the action of o-phthaldehyde (OPA) on the glucansucrase of L. mesenteroides NRRL B-512F in the
presence of T-10 glucan and mild trypsic digestion,
resultant peptides contained lysines which were protected from OPA because of the binding with glucan
[109].
The secondary structure prediction for repeated
units carried out by von Eichel-Streiber et al. [122]
suggested that direct repeating units might possess
the structure of a functional binding pocket. An antiserum directed against a peptide TIDGKKYFN inhibits the cytotoxic e¡ects of C. di¤cile toxin A
[120]. An antiserum directed against a peptide
TGAQTIKGQKLYFKANGQQVKG present in
the C-terminal domain of S. downei Mfe 28 GTF-I
inhibits glucansucrase activity [127]. A 17-kDa peptide coming from mild trypsic digestion of the
C-terminal domain of S. sobrinus 6715 GTF-S was
able to bind with glucan [128].
FEMSRE 642 16-4-99
146
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
The minimum number of these repeated units necessary to ensure glucan binding properties was investigated. For GTF-D, the loss of only one C-terminal
A repeated unit or of two N-terminal A repeated
units is su¤cient to suppress its binding capacity
[91]. This di¡erence tends to support the idea that
all the units are not required in a similar way. Moreover, it appears that the number of required units is
di¡erent for enzymes producing a soluble glucan
than for those producing an insoluble one, the latter
enzymes appearing less sensitive to deletions. The
presence of only the ¢rst two N-terminal repeated
units is su¤cient for GTF-I and GTF-Ia to bind
glucan [21,23,90]. Deletions of three C-terminal units
(A^C) are necessary to a¡ect binding capacity of
GTF-G [129].
5.3. Role of the C-terminal domain in glucansucrase
activity
The presence of the C-terminal glucan binding domain seems to be necessary to keep an active enzyme
[21,23,90,91,115,129]. As for glucan binding capacities, the activity of enzymes producing a soluble glucan appears to be more a¡ected by deletions of the
C-terminal domain than is the activity of enzymes
producing an insoluble one. For GTF-D the loss of
the C-terminal A repeated unit is su¤cient to produce an activity decrease of 90% [91]. The truncation
of the last 85 amino acid residues of the C-terminal
domain of DSR-S resulted in only about 25% of the
initial activity being retained [115]. In contrast, deletion of the three C-terminal units (A^C) of GTF-G is
necessary to produce an activity decrease of 85%
[129]. Only the two N-terminal A repeated units
are necessary to keep GTF-I or GTF-Ia signi¢cantly
active [21,23,90]. The importance of the C-terminal
domain in the glucansucrase catalytic mechanism remains mostly unknown. However, the fact that with
some deleted enzymes, hydrolytic activity remains,
but glucan binding and synthesis properties disappear [23,90], suggests that the C-terminal domain
may be important for the polymer chain growth.
C-Terminal truncations of DSR-S did neither modify
the Km for sucrose, nor the optimum pH and energy
of activation of this enzyme both for the dextran
synthesis reaction and for the oligosaccharide synthesis reaction: it is thus supposed that the C-termi-
nal domain is not directly involved in the catalytic
process for dextran formation, but may make release
of products from the catalytic site easier [115]. The
glucansucrase glucan-binding domain seems also to
be required for the synthesis of low molecular weight
oligosaccharides. C-Terminal truncations of DSR-S
resulted in a strong decrease in oligosaccharide synthesis velocity, whatever the acceptor molecule used
(maltose or fructose) [115]. Moreover, the fact that
the C-terminal domain of DSR-S modulated the size
of oligosaccharides produced in the presence of maltose, without a¡ecting the reaction yield and the activator e¡ect of maltose, seems to show again that its
role may be to facilitate the release of products from
the catalytic site without being directly involved in
the catalysis [115].
The C-terminal domain seems also be involved in
glucan structure determination. Deletion of the three
C-terminal units (A^C) of GTF-G led to an enzyme
being obtained which produced an K(1^6) linked glucan whereas wild-type GTF-G produced a glucan
composed by K(1^6) and K(1^3) glucosidic linkages
[129]. The in-frame fusion of the C-terminal domain
of GTF-B, an enzyme producing an insoluble glucan, with the N-terminal catalytic domain of GTFD, an enzyme producing a soluble glucan, resulted in
the formation of an enzyme producing an insoluble
glucan [130]. However, the inverse in-frame fusion of
the C-terminal domain of GTF-D with the N-terminal catalytic domain of GTF-B did not yield an enzyme producing a soluble glucan [130]. This shows
that the C-terminal domain may in£uence the structure of the produced dextran, but is not the unique
determinant.
6. Conclusions
Glucansucrases were ¢rst identi¢ed more than
50 years ago. They are of industrial value and biologically important due to their key role in the cariogenesis process. Moreover, these enzymes are able to
synthesise oligosaccharides o¡ering large scale applications in various ¢elds and particularly in health
applications. Control of regioselectivity and speci¢city of these enzymes remains the major target with a
view to producing oligosaccharides of structural value [131]. However, the glucansucrase action mecha-
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
nism is still not fully understood. Enzymology studies have enabled two general mechanisms to be proposed involving either one nucleophilic site or two
nucleophilic sites. The understanding of the mechanism of glucansucrase action is complicated by the
fact that many types of products of various structures can be obtained and that glucose coming from
sucrose cleavage can be transferred to the reducing
side of the growing polymer chain, but also to the
non-reducing end of acceptor molecules. Moreover,
numerous aspects of glucansucrase action mechanism, like chain termination or primer action, are
still not well known.
Development of molecular biology techniques has
given further insight into the glucansucrase action
mechanism by allowing several glucansucrase encoding genes to be cloned and structure^function relationship studies to be started. It is clearly shown that
glucansucrases are large-size enzymes presenting a
two domain structure: an N-terminal catalytic domain and a C-terminal glucan binding domain. A
conserved Asp amino acid has been identi¢ed that
makes a covalent link with the glucose moiety coming from sucrose. Site-directed mutagenesis studies
combined with secondary structure prediction studies
have enabled other residues important for enzyme
activity to be identi¢ed, but not their exact role in
the catalytic mechanism of glucansucrases. The degree of involvement of the C-terminal glucan binding
domain in the action mechanism is not fully understood and amino acid residues responsible for binding with dextran have not been identi¢ed.
Comparisons of glucansucrase primary sequences
are not su¤cient for identifying amino acid residues
which may be involved in their mechanisms. Moreover, the lack of crystallographic data does not exclude that single amino acid residue substitution may
cause general conformational changes instead of simple suppression of a functional group. Thus, elucidation of the 3-dimensional structure of glucansucrase
is now the major task in further work. The structure
determination of enzymatic sub-domains can also be
envisaged as their crystallisation should be easier.
This structural information combined with modelisation of enzyme^substrate interactions and with data
coming from kinetic studies obtained with di¡erent
mutants, might lead to the elucidation of the glucansucrase mechanism. However, in order to validate
147
such a mechanism, further investigations, such as
the crystallisation of substrate^enzyme and/or product(s)^enzyme complexes, and the elucidation of the
structure of further glucansucrases, will be necessary.
References
[1] Sidebotham, R.L. (1974). Dextrans. Adv. Carbohydr. Chem.
Biochem. 30, 371^444.
[2] Koepsell, H.J., Tsuchiya, H.M., Hellman, N.N., Kasenko, A.,
Ho¡man, C.A., Sharpe, E.S. and Jackson, R.W. (1953) Enzymatic synthesis of dextran. Acceptor speci¢city and chain initiation. J. Biol. Chem. 200, 793^801.
[3] Seymour, F.R. and Knappk, R.D. (1980) Structural analysis
of dextrans from strains of Leuconostoc and related genera,
that contain 3-O-K-glucosylated-D-glucopyranosyl residues at
the branched points, or in consecutive, linear position. Carbohydr. Res. 81, 105^129.
[4] Hare, M.D., Svensson, S. and Walker, G.J. (1978) Characterization of the extracellular, water-insoluble K-D-glucans of
oral streptococci by methylation analysis, and by enzymic synthesis and degradation. Carbohydr. Res. 66, 254^264.
[5] Groenwall, A.J. and Ingelman, B.J.A. (1948) Manufacture of
infusion and injection £uids. U.S. Patent 2, 437^518.
[6] Garvie, E.I. (1984) Separation of species of the genus Leuconostoc and di¡erentiation of the Leuconostoc from other lactic
acid bacteria, In: Methods in Microbiology (Bergan, T., Ed.),
Vol. 16, pp. 147^178. Academic Press, London.
[7] Soetaert, W., Schwengers, D., Bucholz, K. and Vandamme,
E.J. (1995) A wide range of carbohydrate modi¢cations by a
single microorganism: Leuconostoc mesenteroides. In: Carbohydrate Bioengineering (Petersen, S.B., Svensson, B. and
Pederson, S., Eds.), Vol. 10, pp. 351^358. Elsevier, Amsterdam.
[8] Paul, F., Lopez-Munguia, A., Remaud, M., Pelenc, V. and
Monsan, P. (1992) Method of the production of K-1,2 oligodextrans using Leuconostoc mesenteroides NRRL B-1299. U.S.
patent 5, 141^858.
[9] Djouzi, Z., Andrieux, C., Pelenc, V., Somarriba, S., Popot, F.,
Paul, F., Monsan, P. and Szylit, O. (1995) Degradation and
fermentation of K-glucooligosaccharides by bacterial strains
from human colon : in vitro and in vivo studies in gnotobiotic
rats. J. Appl. Bacteriol. 79, 117^127.
[10] Hamada, S. and Slade, H.D. (1980) Biology, immunology,
and cariogenicity of Streptococcus mutans. Microbiol. Rev.
44, 331^384.
[11] Loesche, W.J. (1986) Role of Streptococcus mutans in human
dental decay. Microbiol. Rev. 50, 353^380.
[12] Aoki, H., Shiroza, T., Hayakawa, M., Sato, S. and Kuramitsu, H.K. (1986) Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect.
Immun. 53, 587^594.
[13] Shiroza, T., Ueda, S. and Kuramitsu, H.K. (1987) Sequence
analysis of the gtfB gene from Streptococcus mutans. J. Bacteriol. 169, 4263^4270.
FEMSRE 642 16-4-99
148
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
[14] Hanada, N. and Kuramitsu, H.K. (1988) Isolation and characterization of the Streptococcus mutans gtfC gene, coding for
both soluble and insoluble glucan synthesis. Infect. Immun.
56, 1999^2005.
[15] Ueda, S., Shiroza, T. and Kuramitsu, H.K. (1988) Sequence
analysis of the gtfC gene from Streptococcus mutans GS 5.
Gene 69, 101^109.
[16] Hanada, N. and Kuramitsu, H.K. (1989) Isolation and characterization of the Streptococcus mutans gtfD gene, coding for
primer-dependent soluble glucan synthesis. Infect. Immun. 57,
2079^2085.
[17] Honda, O., Kato, C. and Kuramitsu, H.K. (1990) Nucleotide
sequence analysis of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme. J. Gen. Microbiol.
136, 2099^2105.
[18] Pucci, M.J., Jones, K.R., Kuramitsu, H.K. and Macrina, F.L.
(1987) Molecular cloning and characterization of the glucosyltransferase C gene (gtfC) from Streptococcus mutans LM7.
Infect. Immun. 55, 2176^2182.
[19] Gilpin, M.L., Russell, R.R.B. and Morrissey, P. (1985) Cloning and expression of two Streptococcus mutans glucosyltransferases in E. coli K-12. Infect. Immun. 49, 414^416.
[20] Russell, R.R.B., Gilpin, M.L., Musaka, H. and Dougan, G.
(1987) Characterization of glucosyltransferase expressed from
a Streptococcus sobrinus gene cloned in Escherichia coli.
J. Gen. Microbiol. 133, 935^944.
[21] Ferretti, J.J., Gilpin, M.L. and Russell, R.R.B. (1987) Nucleotide sequence of a glucosyltransferase gene from Streptococcus
sobrinus Mfe28. J. Bacteriol. 169, 4271^4278.
[22] Gilmore, K.S., Russell, R.R.B. and Ferretti, J.J. (1990) Analysis of the Streptococcus downei gtfS gene, which speci¢es a
glucosyltransferase that synthesizes soluble glucans. Infect.
Immun. 58, 2452^2458.
[23] Abo, H., Matsumura, T., Kodama, T., Ohta, H., Fukui, K.,
Kato, K. and Kagawa, H. (1991) Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (Water-Insoluble glucan synthetase)
J. Bacteriol. 173, 989^996.
[24] Hanada, N., Yamashita, Y., Shibata, Y., Sato, S., Katayama,
T., Takehara, T. and Inoue, M. (1991) Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase
which produces a high-molecular-weight water-soluble glucan.
Infect. Immun. 59, 3434^3438.
[25] Hanada, N., Isobe, Y., Aizawa, Y., Katayama, T., Sato, S.
and Inoue, M. (1993) Nucleotide sequence analysis of the gtfT
gene from Streptococcus sobrinus OMZ176. Infect Immun. 61,
2096^2103.
[26] Sato, S., Masaku, I., Hanada, N., Aizawa, Y., Isobe, Y. and
Isobe, Y. (1993) DNA sequence of the glucosyltransferase
gene of serotype d of Streptococcus sobrinus. DNA Seq. 4,
19^27.
[27] Pitty, L.J., Gi¡ard, P.M., Gilpin, M.L., Russell, R.R.B. and
Jacques, N.A. (1989) Cloning and expression of glucosyltransferase activities from Streptococcus salivarius. J. Dent. Res. 68,
1681^1682.
[28] Gi¡ard, P.M., Simpson, C.L., Milward, C.P. and Jacques,
N.A. (1991) Molecular characterization of a cluster of at least
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
two glucosyltransferases genes in Streptococcus salivarius
ATCC 25975. J. Gen. Microbiol. 137, 2577^2593.
Simpson, C.L., Cheetham, N.W.H., Gi¡ard, P.M. and Jacques, N.A. (1995) Four glucosyltransferases, GTFJ, GTFK,
GTFL and GTFM from Streptococcus salivarius ATCC
25975. Microbiology 141, 1451^1460.
Gi¡ard, P.M., Allen, D.M., Milward, C.P., Simpson, C.L. and
Jacques, N.A. (1993) Sequence of the gtfK gene of Streptococcus salivarius ATCC 25975 and evolution of the gtf genes of
oral streptococci. J. Gen. Microbiol. 139, 1511^1522.
Banas, J.A., Simon, D., Williams, L.K., Ferretti, J.J. and
Russell, R.R.B. (1994) Analysis of a primer-independent
GTF-I from Streptococcus salivarius. FEMS Microbiol. Lett.
123, 349^354.
Simpson, C.L., Gi¡ard, P.M. and Jacques, N.A. (1995) Streptococcus salivarius ATCC 25975 possesses at least two genes
coding for primer independent glucosyltransferases. Infect.
Immun. 63, 609^621.
Vickermann, M.M., Sulavik, M.C., Nowak, J.D., Gardner,
N.M., Jones, C.W. and Clewell, D.B. (1997) Nucleotide sequence analysis of the Streptococcus gordonii glucosyltransferase gene, gtfG. DNA Seq. 7, 83^95.
Wilke-Douglas, M., Perchorowicz, J.T., Houck, C.M. and
Thomas, B.R. (1989) Methods and compositions for altering
physical characteristics of fruit and fruit products. PCT patent
WO 89/12386.
Monchois, V., Willemot, R.M., Remaud-Simeon, M., Croux,
C. and Monsan, P. (1996) Cloning and sequencing of a gene
coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only K(1^6) and K(1^3) linkages. Gene 182, 23^32.
Monchois, V., Remaud-Simeon, Monsan, P. and Willemot,
R.M. (1998) Cloning and sequencing of an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL
B-1299 synthesizing only K(1^6) glucan. FEMS Microbiol.
Lett. 159, 307^315.
Mooser, G. (1992) Glycosidases and glycosyltransferases. The
Enzymes. Vol XX, pp. 187^221. Academic Press, New York.
Ebert, K.H. and Schenk, G. (1968) Mechanism of biopolymer
growth: the formation of dextran and levan. Adv. Enzymol.
30, 179^210.
Robyt, J.F., Kimble, B.K. and Walseth, T.F. (1974) The
mechanism of dextransucrase action. Direction of dextran biosynthesis. Arch. Biochem. Biophys. 165, 634^640.
Luzio, G.A. and Mayer, R.M. (1983) The hydrolysis of sucrose by dextransucrase. Carbohydr. Res. 111, 311^318.
Mooser, G. and Iwaoka, K.R. (1989) Sucrose 6-K-glucosyltransferase from Streptococcus sobrinus: characterization of
a glucosyl^enzyme complex. Biochemistry 28, 443^449.
Mayer, R.M. (1987) Dextransucrase: a glucosyltransferase
from Streptococcus sanguis. Methods Enzymol. 138, 649^661.
Hehre, E.J. (1941) Comparison of dextran synthesis by Leuconostoc enzyme with starch synthesis with potato phosphorylase. Proc. Soc. Exp. Biol. Med. 54, 240^241.
Hestrin, S., Avireni-Shapiro, S. and Aschner, M. (1943) The
enzymatic production of levan. Biochemistry 37, 450^456.
Bovey, F.A. (1959) Enzymatic polymerisation I. Molecular
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
weight and branching during the formation of dextran.
J. Polymer Sci. 35, 167^182.
Tsuchiya, H.M., Hellman, N.N. and Koepsell, H.J. (1953)
Factors a¡ecting molecular weight of enzymatically synthesized dextran. J. Am. Chem. Soc. 75, 757^758.
Kobayashi, M., Yokoyama, I. and Matsuda, K. (1984) Activation of dextransucrase from Leuconostoc mesenteroides by
the substrate dextran. Agric. Biol. Chem. 48, 221^223.
Kobayashi, M., Yokoyama, I. and Matsuda, K. (1985) E¡ectors di¡erently modulating the dextransucrase activity of Leuconostoc mesenteroides. Agric. Biol. Chem. 49, 3189^3195.
Kobayashi, M., Mihara, M. and Matsuda, K. (1986) Dextransucrase from Leuconostoc mesenteroides NRRL B-512F: characterization of the enzyme bound to sephadex gel. Agric. Biol.
Chem. 50, 551^557.
Kobayashi, M., Yokoyama, I. and Matsuda, K. (1986) Substrate binding sites of Leuconostoc dextransucrase evaluated
by inhibition kinetics. Agric. Biol. Chem. 50, 2585^2590.
Yokoyama, I., Kobayashi, M. and Matsuda, K. (1985) Comparison of the multplicity of dextransucrases from six strains
of Leuconostoc mesenteroides. Agric. Biol. Chem. 49, 501^507.
Germaine, G.R., Chludzinski, A.M. and Schachtele, C.F.
(1974) Streptococcus mutans dextransucrase: requirement for
primer dextran. J. Bacteriol. 120, 287^294.
Germaine, G.R., Harlander, S.K., Leung, W.L.S. and Schachtele, C.F. (1977) Streptococcus mutans : functioning of primer
dextran and endogenous dextranases in water-soluble and
water-insoluble glucan synthesis. Infect. Immun. 16, 637^648.
Robyt, J.F. and Corrigan, A.J. (1977) The mechanism of dextransucrase action. Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modi¢ed dextran
and the non existence of the primer requirement for the synthesis of dextran. Arch. Biochem. Biophys. 183, 726^731.
Robyt, J.F., Kim, D. and Yu, L. (1995) Mechanism of dextran activation of dextransucrase. Carbohydr. Res. 266, 293^
299.
Mooser, G., Shur, D., Lyou, M. and Watanabe, C. (1985)
Kinetic studies on dextransucrase from the cariogenic oral
bacterium Streptococcus mutans. J. Biol. Chem. 260, 6907^
6915.
Sinnot, M.L. (1990) Catalytic mechanisms of enzymic glycosyl
transfer. Chem. Rev. 90, 1171^1202.
Su, D.L. and Robyt, J.F. (1994) Determination of the number
of sucrose and acceptor binding sites for Leuconostoc mesenteroides B-512FM dextransucrase, and the con¢rmation of the
two site mechanism for dextran synthesis. Arch. Biochem.
Biophys. 308, 471^476.
Robyt, J.F. and Martin, P.J. (1983) Mechanism of synthesis of
D-glucans by D-glucosyltransferases from Streptococcus mutans
6715. Carbohydr. Res. 113, 301^315.
Ditson, S.L. and Mayer, R.M. (1984) Dextransucrase : the
direction of chain growth during autopolymerisation. Carbohydr. Res. 126, 170^175.
Germaine, G.R. and Schachtele, C.F. (1976) Streptococcus
mutans dextransucrase : mode of interaction with high molecular-weight dextran and role in cellular aggregation. Infect.
Immun. 13, 365^372.
149
[62] Koba, S.F. and Mayer, R.M. (1991) Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl K-Dglucopyranoside. Carbohydr. Res. 211, 317^326.
[63] Mazza, J.C., Akjerman, A. and Edwards, J.R. (1975) Synthesis of K-D-glycopyranosyl K-L-sorbofuranoside and its use as a
D-glucosyl donor. Carbohydr. Res. 40, 402^406.
[64] Figures, W.R. and Edwards, J.R. (1976) K-D-Glucopyranosyl
£uoride as a D-glucopyranosyl donor for a glycosyltransferase
complex from Streptococcus mutans FA1. Carbohydr. Res. 48,
245^253.
[65] Binder, T.P. and Robyt, J.F. (1983) p-Nitrophenyl K-D-glucopyranoside, a new substrate to glucansucrases. Carbohydr.
Res. 124, 287^299.
[66] Robyt, J.F. and Elkund, S.H. (1983) Relative, quantitative
e¡ects of acceptors in the reaction of Leuconostoc mesenteroides NRRL B-512F dextransucrase. Carbohydr. Res. 68, 95^
111.
[67] Yamauchi, F. and Ohawada, Y. (1969) Synthesis of oligosaccharides by growing culture of Leuconostoc mesenteroides.
Part IV. Oligosaccharide formation in the presence of various
types of glucobioses as acceptors. Agric. Biol. Chem. 33,
1295^1300.
[68] Robyt, J.F. and Walseth, T.F. (1978) The mechanism of acceptor reactions of Leuconostoc mesenteroides NRRL B-512F
dextransucrase. Carbohydr. Res. 61, 433^435.
[69] Stolada, F.H., Sharpe, E.H. and Koepsell, H.J. (1956) The
preparation, properties and structure of the disaccharide leucrose. J. Am. Chem. Soc. 78, 2514^2518.
[70] Tanrivseven, A. and Robyt, J.F. (1992) Inhibition of dextran
synthesis by acceptor reactions of dextransucrase, and the
demonstration of a separate acceptor binding site. Carbohydr.
Res. 225, 321^329.
[71] Kobayashi, M. and Matsuda, K. (1978) Inhibition of dextran
synthesis by glucoamylase and endodextranase. Carbohydr.
Res. 66, 277^288.
[72] Paul, F., Oriol, E., Auriol, D. and Monsan, P. (1986) Acceptor reaction of a highly puri¢ed dextransucrase with maltose
and oligosaccharides. Application to the synthesis of controlled-molecular-weight dextrans. Carbohydr. Res. 149,
433^441.
[73] Mayer, R.M., Matthews, M.M., Futerman, C.L., Parnaik,
V.K. and Jung, S.M. (1981) Dextransucrase : acceptor substrate reactions. Arch. Biochem. Biophys. 208, 278^287.
[74] Boker, M., Jordening, H. and Buchholz, K. (1994) Kinetics of
leucrose formation from sucrose by dextransucrase. Biotechnol. Bioeng. 43, 392^394.
[75] Figures, W.R. and Edwards, J.R. (1981) K-D-Glucosyltransferase of Streptococcus mutans : isolation of two forms of the
enzyme that bind the insoluble dextran. Carbohydr. Res. 88,
107^117.
[76] Robyt, J.F. and Taniguchi, H. (1976) The mechanism of dextransucrase action. Biosynthesis of branch linkages by acceptor reactions with dextran. Arch. Biochem. Biophys. 174, 129^
135.
[77] McCabe, W.L. and Smith, E.E. (1978) The dextran acceptor
reactions of dextransucrase from Streptococcus mutans K1-R.
Carbohydr. Res. 63, 223^239.
FEMSRE 642 16-4-99
150
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
[78] Musaka, H., Shimamura, A. and Tsumori, H. (1979) E¡ects
of salts on water insoluble glucan formation by glucosyltransferase of Streptococcus mutans. Infect. Immun. 23, 564^
570.
[79] Newman, B.M., White, P., Mohan, S.B. and Cole, J.A. (1980)
E¡ects of dextran and ammonium sulfate on the reaction catalysed by glucosyltransferase complex from Streptococcus mutans. J. Gen. Microbiol. 118, 353^356.
[80] Sato, S., Koga, T., Yakushill, T., Nagasawa, S. and Inoue, M.
(1982) E¡ect of exogenous soluble dextrans on insoluble glucan synthesis by Streptococcus mutans glucosyltransferase. Microbiology 34, 99^112.
[81] Ebert, K.H. and Brosche, M. (1967) Origin of branches in
native dextrans. Biopolymers 5, 423^430.
[82] Kim, D. and Robyt, J.F. (1996) Dextransucrase constitutive
mutants of Leuconostoc mesenteroides NRRL B-1299. Enzyme
Microbiol. Technol. 17, 1050^1056.
[83] Cote, G.L. and Robyt, J.F. (1984) The formation of K-D-(1^3)
branch linkages by K-D-glucansucrase from S. mutans 6715
producing a soluble D-glucan. Carbohydr. Res. 127, 95^
107.
[84] Monchois, V., Remaud-Simeon, M., Russell, R.R.B., Monsan, P. and Willemot, R.M. (1997) Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identi¢cation of amino acid residues playing a key role
in enzyme activity. Appl. Microbiol. Biotechnol. 48, 465^472.
[85] Russell, R.R.B. (1990) Molecular genetics of glucan metabolism in oral Streptococci. Arch. Oral. Biol. 35, 53S^58S.
[86] Izard, J.W. and Kendall, D.A. (1994) Signal peptides : exquisitely designed transport promoters. Mol. Microbiol. 13, 765^
773.
[87] Shiroza, T. and Kuramitsu, H.K. (1988) Sequence analysis of
the Streptococcus mutans fructosyltransferase gene and £anking regions. J. Bacteriol. 170, 4263^4270.
[88] Mooser, G. and Wong, C. (1988) Isolation of glucan-binding
domain of glucosyltransferase (1,6-K-glucan synthase) from
Streptococcus sobrinus. Infect. Immun. 56, 880^884.
[89] Kobayashi, S., Koga, K., Hayashida, O., Nakano, Y. and
Hasegawa, Y. (1989) Glucan-binding domain of a glucosyltransferase from Streptococcus sobrinus: isolation of a 55-kilodalton peptide trypsin digest of glucosyltransferase prebound
to insoluble glucan. Infect. Immun. 57, 2210^2213.
[90] Kato, C. and Kuramitsu, H.K. (1990) Carboxy-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I
enzyme. FEMS Microbiol. Lett. 72, 299^302.
[91] Lis, M., Shiroza, T. and Kuramitsu, H.K. (1995) Role of the
C-terminal direct repeating units of the Streptococcus mutans
glucosyltransferase-S in glucan binding. Appl. Env. Microbiol.
61, 2040^2042.
[92] Teeri, T.T., Koivula, A., Linder, M., Reinikainen, T., Ruohonen, L., Srisodsuk, M., Claeyssens, M. and Alwyn Jones, T.
(1995) Modes of action of two Trichoderma reesei cellobiohydrolases. Progr. Biotechnol. 10, 211^224.
[93] MacGregor, A.E., Jespersen, H.M. and Svensson, B. (1996) A
circularly permuted K-amylase type K/L barrel structure in
glucan synthesizing glucosyltransferases. FEBS Lett. 378,
263^266.
[94] Devulapalle, K.S., Goodman, S.D., Gao, Q., Hemsley, A.
and Mooser, G. (1997) Knowledge-base model of glucosyltransferase from the oral bacteria group of mutans streptococci. Protein Sci. 6, 2489^2493.
[95] Mooser, G., Hefta, S.A., Paxton, R.J., Shively, J.E. and Lee,
T.D. (1991) Isolation and sequence of an active-site peptide
containing a catalytic aspartic acid from two Streptococcus
sobrinus glucosyltransferases. J. Biol. Chem. 266, 8916^8922.
[96] Kato, C., Nakano, Y., Lis, M. and Kuramitsu, H.K. (1992)
Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases. Biochem. Biophys. Res.
Commun. 189, 1184^1188.
[97] Cope, P.A. and Mooser, G. (1993) Antibodies against activesite peptides common to glucosyltransferases of mutans
Streptococci. Infect. Immun. 61, 4814^4817.
[98] Laloi, P., Munro, C.L., Jones, K.R. and Macrina, F.L.
(1996) Immunologic characteristics of a Streptococcus mutans
glucosyltransferase B sucrose binding site peptide-cholera
toxin B-subunit chimeric protein. Infect. Immun. 64, 28^36.
[99] Smith, D.J., Taubman, M.A., King, W.F, Eida, S., Powell,
J.R. and Eastcott, J. (1994) Immunological characteristics of
a synthetic peptide associated with a catalytic domain of
mutans streptococcal glucosyltransferase. Infect. Immun.
62, 5470^5476.
[100] Taubman, M.A., Holmberg, C.J. and Smith, D.J. (1995) Immunization of rats with synthetic peptide constructs from the
glucan-binding or catalytic region of mutans streptococcal
glucosyltransferases protects against dental caries. Infect. Immun. 63, 3088^3093.
[101] Koga, T. and Inoue, M. (1981) Inactivation of D-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation. Carbohydr. Res. 93,
125^133.
[102] Fu, D. and Robyt, J.F. (1988) Essential histidine residues in
dextransucrase : chemical modi¢cation by diethyl pyrocarbonate and dye photo-oxidation. Carbohydr. Res. 183, 97^
109.
[103] Funane, K., Shiraiwa, M., Hashimoto, K., Ichishima, E. and
Kobayashi, M. (1993) An active-site peptide containing the
second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modi¢cations. Biochemistry 32, 13696^13702.
[104] Tsumori, H., Minami, T. and Kuramitsu, H.K. (1997) Identi¢cation of essential amino acids in the Streptococcus mutans
glucosyltransferases. J. Bacteriol. 179, 3391^3396.
[105] Chia, J.S., Hsu, T.Y., Teng, L.J., Chen, J.Y., Hahn, L.J. and
Yang, C.S. (1991) Glucosyltransferase gene polymorphism
among Streptococcus mutans strains. Infect. Immun. 59,
1656^1660.
[106] Chia, J.S., Lin, S.W., Hsu, T.Y., Chen, J.Y., Kwan, H.W.
and Yang, C.S. (1993) Analysis of a DNA polymorphic region in the gtfB and gtfC genes of Streptococcus mutans.
Infect. Immun. 61, 1563^1566.
[107] Chia, J.S., Lin, R.H., Lin, S.W., Chen, J.Y. and Yang, C.S.
(1993) Inhibition of glucosyltransferase activities of Streptococcus mutans by a monoclonal antibody to a subsequence
peptide. Infect. Immun. 61, 4686^4695.
FEMSRE 642 16-4-99
V. Monchois et al. / FEMS Microbiology Reviews 23 (1999) 131^151
[108] Dertzbaugh, M.T. and Macrina, F.L. (1990) Inhibition of
Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide. Infect. Immun. 58, 1509^1513.
[109] Funane, K., Arai, T., Chiba, Y., Hashimoto, K., Ichishima,
E. and Kobayashi, M. (1995) Sucrose and dextran-binding
sites of dextransucrase analyzed by chemical modi¢cations
with o-phthalaldehyde. Oyo Tos. Kaga. 42, 27^35.
[110] Goyal, A. and Katiyar, S.S. (1995) Involvement of a lysine
residue in the inactivation of Leuconostoc mesenteroides
NRRL B-512F dextransucrase by o-phthalaldehyde. Biochem. Mol. Biol. Int. 36, 579^585.
[111] Goyal, A. and Katiyar, S.S. (1995) 2,4,6 Trinitrobenzenesulphonic acid as a probe for lysine at the active site of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.
Biochem. Mol. Biol. Int. 36, 639^647.
[112] Shimamura, A., Nakano, Y.J., Musaka, H. and Kuramitsu,
H.K. (1994) Identi¢cation of amino acid residues in Streptococcus mutans glucosyltransferases in£uencing the structure
of the glucan product. J. Bacteriol. 176, 4845^4850.
[113] Jespersen, H.M., MacGregor, A.E., Henrissat, B., Sierk,
M.R. and Svensson, B. (1993) Starch- and glycogen-debranching and branching enzymes : prediction of structural
features of the catalytic (L/K)8 -barrel domain and evolutionary relationship to other amylolytic enzymes. J. Protein
Chem. 12, 791^805.
[114] Farber, C.K. (1993) An K/L barrell full of evolutionary trouble. Curr. Opin. Struct. Biol. 3, 409^412.
[115] Monchois, V., Reverte, A., Remaud-Simeon, H., Monsan, P.
and Willemot, R.M. (1998) E¡ect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis. Appl. Environ. Microbiol. 64, 1649.
[116] Banas, J.A., Russell, R.R.B. and Ferretti, J.J. (1990) Sequence analysis of the gene for the glucan-binding protein
of Streptococcus mutans Ingbritt. Infect. Immun. 58, 667^
673.
[117] Sun, J.W., Wanda, S.Y., Camilli, A. and Curtiss III, R.
(1994) Cloning and DNA sequencing of the dextranse inhibitor gene (dei) from Streptococcus sobrinus. J. Bacteriol. 176,
7213^7222.
[118] Sun, J.W., Wanda, S.Y. and Curtiss III, R. (1995) Puri¢cation, characterization and speci¢city of dextranase inhibitor
(Dei) expressed from Streptococcus sobrinus UAB108 gene
cloned in Escherichia coli. J. Bacteriol. 177, 1703^1711.
[119] Wren, B.W. (1991) A family of clostridial and streptococcal
ligand-binding proteins with conserved C-terminal repeat sequences. Mol. Microbiol. 5, 797^803.
151
[120] Wren, B.W., Russell, R.R.B. and Tabaqchali, S. (1991) Antigenic cross-reactivity and functional inhibition by antibodies
to Clostridium di¤cile toxin A, Streptococcus mutans glucanbinding protein and a synthetic peptide. Infect. Immun. 59,
3151^3155.
[121] Tucker, K.D. and Wilkins, T.D. (1991) Toxin A of Clostridium di¤cile binds to the human carbohydrate antigens I, X,
and Y. Infect. Immun. 59, 561^564.
[122] von Eichel-Streiber, C., Saueborn, M. and Kuramitsu, H.K.
(1992) Evidence for a modular structure of the homologous
repetitive C-terminal carbohydrate-binding sites of Clostridium di¤cile toxins and Streptococcus mutans glucosyltransferases. J. Bacteriol. 174, 6707^6710.
[123] Gi¡ard, P.M. and Jacques, N.A. (1994) De¢nition of a fundamental repeating unit in streptococcal glucosyltransferase
glucan-binding regions and related sequences. J. Dent. Res.
73, 1133^1141.
[124] Quiocho, F.A. (1986) Carbohydrate-binding proteins : tertiary structures and protein^sugar interactions. Annu. Rev.
Biochem. 55, 287^315.
[125] Lemieux, R.U. (1989) The origin of the speci¢city in recognition of oligosaccharides by proteins. Chem. Soc. Rev. 18,
347^374.
[126] Singh, J.S., Taylor, K.J. and Doyle, R.J. (1993) Essential
amino acids involved in glucan-dependent aggregation of
Streptococcus sobrinus. Carbohydr. Res. 244, 137^147.
[127] Smith, D.J., Taubman, M.A., Holmberg, C.F., Eastcott, J.,
King, W.J. and Ali-Salaam, P. (1993) Antigenicity and immunogenicity of a synthetic peptide derived from a glucanbinding domain of mutans streptococcal glucosyltransferase.
Infect. Immun. 61, 2899^2905.
[128] Wong, C., Hefta, S.A., Paxton, R.J., Shively, J.E. and
Mooser, G. (1990) Size and subdomain architecture of the
glucan-binding domain of sucrose : 3-K-D glucosyltransferase from Streptococcus sobrinus. Infect. Immun. 58, 2165^
2170.
[129] Vickermann, M.M., Sulavik, M.C., Minick, P.E. and Clewell, D.B. (1996) Changes in the carboxy-terminal repeat
region a¡ect extracellular activity and glucan products of
Streptococcus gordonii glucosyltransferase. Infect. Immun.
64, 5117^5128.
[130] Nakano, Y.J. and Kuramitsu, H.K. (1992) Mechanism of
Streptococcus mutans glucosyltransferases : hybrid^enzyme
analysis. J. Bacteriol. 174, 5639^5646.
[131] Monsan, P. and Paul, F. (1995) Enzymatic synthesis of oligosaccharides. FEMS Microbiol. Rev. 16, 187.
FEMSRE 642 16-4-99