Alternansucrase acceptor reactions with D-tagatose and L-glucose

Article abstract of DOI:10.1016/j.carres.2004.11.013

Alternansucrase (EC 2.4.1.140) is a d-glucansucrase that synthesizes an alternating α-(1→3), (1→6)-linked d-glucan from sucrose. It also synthesizes oligosaccharides via d-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient monosaccharide acceptors are d-tagatose and l-glucose. In the presence of d-tagatose, alternansucrase produced the disaccharide α-d-glucopyranosyl-(1→1)-β-d-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable to isolate a disaccharide product from l-glucose, but the trisaccharide α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-(1→4) -l-glucose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end d-glucose residue has been replaced by its l-enantiomer. The putative l-glucose disaccharide product, produced by glucoamylase hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, α-d-glucopyranosyl- (1→4)-l-glucose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of α-d-glucopyranosyl-(1→4)-l-glucose and maltose was performed through computer modeling to identify common features, which may be important in acceptor affinity by alternansucrase.

Full text of DOI:10.1016/j.carres.2004.11.013

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 257–262
Alternansucrase acceptor reactions with D-tagatose and L-glucose
a,
ˆ ´
Gregory L. Cote, Christopher A. Dunlap, Michael Appell and FrankA. Momany
b
c
c
*
aBioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service,
United States Department of Agriculture , 1815North University Street, Peoria, IL 61604, USA
bCrop Bioprotection, National Center for Agricultural Utilization Research, Agricultural Research Service,
United States Department of Agriculture , 1815North University Street, Peoria, IL 61604, USA
cPlant Polymer Research Units, National Center for Agricultural Utilization Research, Agricultural Research Service,
United States Department of Agriculture , 1815North University Street, Peoria, IL 61604, USA
Received 20 September 2004; accepted 15 November 2004
Available online 18 December 2004
Abstract—Alternansucrase (EC 2.4.1.140) is a D-glucansucrase that synthesizes an alternating a-(1!3), (1!6)-linked D-glucan from
sucrose. It also synthesizes oligosaccharides via D-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient
monosaccharide acceptors are D-tagatose and L-glucose. In the presence of D-tagatose, alternansucrase produced the disaccharide
a-D-glucopyranosyl-(1!1)-b-D-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable
to isolate a disaccharide product from L-glucose, but the trisaccharide a-D-glucopyranosyl-(1!6)-a-D-glucopyranosyl-(1!4)-L-glu-
cose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end
D-glucose residue has been replaced by its L-enantiomer. The putative L-glucose disaccharide product, produced by glucoamylase
hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, a-D-glucopyranosyl-(1!4)-L-glu-
cose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of a-D-
glucopyranosyl-(1!4)-L-glucose and maltose was performed through computer modeling to identify common features, which may
be important in acceptor affinity by alternansucrase.
Published by Elsevier Ltd.
Keywords: Alternansucrase; Glucansucrase; Acceptor reactions; D-Tagatose; L-Glucose
1. Introduction
gard. Not only do its acceptor products differ in structure
from those synthesized by Leuconostoc mesenteroides
NRRL B-512F dextransucrase,2,3 but they are also pro-
duced somewhat more efficiently.3 These oligosaccharide
acceptor products may be useful as prebiotics.4
In this study, we describe the structures of alternan-
sucrase acceptor products arising from D-tagatose and
from L-glucose. Our previous workhas shown that both
of these compounds are among the more efficient mono-
saccharide acceptors for alternansucrase.4 D-Tagatose is
of interest due to its recent approval as an alternative
sweetener in foods5 and its similarity to D-fructose, from
which it differs only by inversion of the 4-OH group. As
it becomes more readily available, it may represent
a useful material for the synthesis of functional
oligosaccharides.
Glucansucrases are D-glucosyltransferases that synthe-
size a-D-glucans from sucrose. They are of interest not
only for their ability to synthesize unique polysaccha-
rides, but also for their ability to synthesize oligosaccha-
rides via acceptor reaction. Alternansucrase (EC
2.4.1.140), which synthesizes an alternating a-(1!3),
(1!6)-linked D-glucan1 is especially interesting in this re-
*
Corresponding author. Tel.: +1 309 681 6319; fax: +1 309 681 6040;
e-mail: cotegl@ncaur.usda.gov
Mention of trade names or commercial products in this article is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture.
0008-6215/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.carres.2004.11.013

ˆ ´
G. L. Cote et al. / Carbohydrate Research 340 (2005) 257–262
258
2. Experimental
2.4. Molecular modeling
2.1. Materials
All calculations reported here were carried out on Paral-
lel Quantum Solutions hardware and software (v3.1,
PQS Ab Initio Program Package, Parallel Quantum
Solutions, 2013 Green Acres, Suite E, Fayetteville, AR
72703). Geometry optimizations were carried out at
the B3LYP/6-31+G* and B3LYP/6-311++G** levels as
previously described with mentioned modifications.10
Geometry optimization was considered satisfactory if
the energy differences between cycles was less than
1 · 10À6 Hartree and a gradient of 3 · 10À4 a.u. All re-
sults reported here are at the B3LYP/6-311++G** level.
Zero-point vibrational energy, enthalpy, and entropy
were calculated using an analytical hessian program
with the default threshold. Models were initially built
and displayed using Hyperchem Professional Release
7.5 for Windows on a Dell 8200 PC (HyperChem 7.5,
Hypercube, Inc., 1115 NW 4th Street, Gainesville, FL
32601).
L-Glucose, sucrose and Rhizopus sp. glucoamylase were
purchased from Sigma–Aldrich Co. (St. Louis, MO,
USA). Glucoamylase was dialyzed against water prior
to use in order to remove low-MW contaminants.
D-Tagatose was a gift from Arla Foods Ingredients
(Union, NJ, USA).
2.2. Acceptor reaction conditions
Acceptor reactions were carried out essentially as previ-
ously described,2,4 using alternansucrase from Leuconos-
toc mesenteroides NRRL B-21297. Dialyzed and
concentrated cell-free culture fluid was used as alternan-
sucrase without further purification, as this strain pro-
duces only alternansucrase with no detectable levels of
dextransucrase.6 Variations in the concentrations of
sucrose and acceptor affect the distribution of acceptor
products, but do not influence their structures.7
3. Results and discussion
3.1. D-Tagatose
2.3. Analytical and preparative methods
Oligosaccharide products were isolated from reaction
mixtures by ion-exchange and gel-permeation chroma-
tography. Mixtures were initially chromatographed over
Dowex 99CA/320 ion-exchange resin (Ca2+ form) in
water. This successfully removed fructose and leucrose
from the other components and partially resolved
lower-DP (degree of polymerization) oligosaccharides.
Appropriate fractions were then chromatographed over
Bio-Gel P-2 (Bio-Rad, Hercules, CA, USA) to isolate
oligosaccharides based on DP. Analytical thin-layer
chromatography was performed on Whatman K5 silica
gel plates, using multiple ascents in 4:1 acetonitrile–
water as previously described.2,8 Quantitative densito-
metry was performed on scanned images of the TLC
plates by use of Un-Scan-It gel software (SilkScientific,
Orem, UT, USA). Structures were determined by meth-
ylation/GC–MS and NMR spectroscopy.9 1H, 13C,
COSY, HMQC, and HMBC NMR spectra were re-
corded for each sample on a Bruker 400 MHz instru-
ment in D2O at 27 ꢀC. Peakassignments for NMR
spectra were made based on the 2D NMR experiments,
and the glycosidic linkages were confirmed by HMBC
long-range coupling experiments.
Alternansucrase reaction mixtures with sucrose and
D-tagatose were first chromatographed over Dowex
99CA/320 to remove fructose. Leucrose was separated
from the disaccharide acceptor product by two passages
over Bio-Gel P-2; apparently their hydrodynamic radii
differ sufficiently to enable this separation.
Upon borohydride reduction and subsequent methyl-
ation, the disaccharide product yielded only 2,3,4,6-
tetra-O-methylhexitol derivatives, which were not
resolved by gas–liquid chromatography. This suggested
a 1,1-linked disaccharide. The structure was determined
to be a-D-glucopyranosyl-(1!1)-b-D-tagatopyranose
(structure 1) by NMR spectroscopy. Peakassignments
appear in Table 1. It is noteworthy that the NMR spec-
trum reveals only a single anomeric form of this disac-
charide in aqueous solution. A similar phenomenon
has been noted for the a-D-glucopyranosyl D-fructose
disaccharides trehalulose11 and leucrose, in which the
D-fructosyl moiety occurs predominantly in the b-pyr-
anosyl form.
In the presence of sucrose and D-tagatose, alternan-
sucrase catalyzes three competing reactions. The first is
Table 1. NMR peakassignments for disaccharide acceptor product 1 from D-tagatose
H-1a
H-1b
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6a
C-6
H-6b
b-D-Tagp
a-D-Glcp
3.32
3.81
69.17
4.87
—
3.75
3.76
3.8
3.5
3.68
3.74
97.82
3.43
71.42
66.16
3.63
72.94
70.79
3.31
69.39
70.05
3.59
71.84
62.25
3.61
60.37
98.32

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G. L. Cote et al. / Carbohydrate Research 340 (2005) 257–262
259
Fructose
Disaccharide acceptor product
Origin
L-Glc
D-Tag
Leucrose
Trisaccharide acceptor products
500
1000
1500
Migration distance (pixels)
Figure 1. TLC densitogram of D-tagatose (- - - -) and L-glucose (—–) acceptor reaction mixtures. Initial monosaccharide acceptor concn was 0.22 M,
initial sucrose concn was 0.47 M, and enzyme concn was 0.2 U/mL in 50 mM pH 5.4 NaOAc buffer with 0.02% NaN3, carried out at 20 ꢀC for 48 h.
polymerization of the D-glucosyl moiety of sucrose to
yield alternan.1 The second and third are the acceptor
reactions consisting of D-glucopyranosyl transfer to
either D-tagatose or to the D-fructose liberated from su-
crose. The latter reaction yields leucrose as the primary
product.2 It is well established that the relative amounts
of alternan, leucrose, and other acceptor products de-
pend on the concentrations of sucrose and acceptor.
To measure these parameters for the D-tagatose reac-
tion, acceptor reactions were carried out at various sub-
strate concentrations and product yields were measured
by TLC densitometry. Figure 1 shows a typical TLC
densitogram of both a D-tagatose and an L-glucose
acceptor reaction mixture. Results of the quantitative
analyses appear in Figures 2 and 3. At sufficiently high
concentrations of acceptor, up to 94% of the glucosyl
portion of sucrose was incorporated into oligosaccha-
ride acceptor products. Within the concentration range
tested here, the best ratio of disaccharide product to leu-
crose occurred at a sucrose concentration of approxi-
mately 0.2 M and a D-tagatose concentration of
1.25 M. It may be assumed that at lower sucrose concen-
trations and higher acceptor concentrations, the prod-
uct/leucrose ratio would be even more favorable, as
the lower sucrose concentrations would yield lower fruc-
tose concentrations. However, since D-tagatose is more
expensive than sucrose, it would be best to optimize con-
centrations to maximize yields from D-tagatose, rather
than from sucrose, while still minimizing leucrose yields.
Figure 2. Percent conversion of sucrose to oligosaccharides as a
function of sucrose and D-tagatose concentrations.
Figure 3. Ratio of disaccharide 1 to leucrose in product mixture as a
function of sucrose and D-tagatose concentrations.

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G. L. Cote et al. / Carbohydrate Research 340 (2005) 257–262
260
This would most likely occur at both high sucrose and
high acceptor concentrations (Figs. 2 and 3).
Other, higher-DP acceptor products were also
formed, but were not isolated. It is presumed that these
arise from subsequent D-glucopyranosyl transfers to the
D-glucopyranosyl moiety of the disaccharide product,
and so their structures should be analogous to those pre-
viously described.1,4
glucopyranosyl-(1!4)-L-glucose, is initially formed,
but is such a good acceptor that it immediately gives
rise to the trisaccharide. To test this hypothesis, an
aqueous solution of trisaccharide 2 was treated with
an excess of dialyzed Rhizopus sp. glucoamylase in
water at room temperature until no trisaccharide re-
mained, as judged by TLC (reaction time = 5 days).
The products were glucose and an unknown disaccha-
ride. The latter was isolated from this mixture by Bio-
3.2. L-Glucose
Thin-layer chromatographic analysis of alternansucrase
acceptor reactions invariably showed only a single disac-
charide product was formed in measurable quantities at
all stages of the reaction that we were able to sample
(e.g., Fig. 1). Isolation of this disaccharide showed it
to be leucrose.
OH
O
HO
HO
OH
O
OH
O
HO
However, significant yields of a trisaccharide and les-
ser amounts of higher-DP products were formed, and
the trisaccharide was readily isolated by Bio-Gel P-2
chromatography. Methylation analysis and GC–MS
revealed equimolar amounts of 2,3,4,6-tetra-O-methyl-
glucose, 2,3,4-tri-O-methylglucose and 2,3,6-tri-O-
methylglucose, indicating a trisaccharide with a (1!6)-
and a (1!4)-linkage. Analysis of the NMR spectra
showed the compound to be a-D-glucopyranosyl-
(1!6)-a-D-glucopyranosyl-(1!4)-L-glucose (structure
2, Table 2).
HO
HO
1
OH
O
HO
HO
OH
O
O
HO
HO
OH
HO
O
OH
It is difficult to understand why no disaccharide
acceptor product was observed in the L-glucose reaction
mixtures. One hypothesis is that the disaccharide, a-D-
O
H, OH
HO
2
Table 2. NMR peakassignments for acceptor products from L-glucose
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6a
C-6
H6b
Disaccharidea
a-L-Glcp
5.15
91.84
4.92
3.48
71.50
3.49
3.90
70.50
3.61
3.50
77.65
3.45
3.53
71.50
3.95
3.77
60.10
3.72
3.91
3.97
3.92
3.97
a-D-Glcp-(1!4)
b-L-Glcp
99.42
4.56
71.50
3.22
72.84
3.54
69.25
3.53
71.80
3.53
60.10
3.79
95.76
4.92
99.46
74.50
3.49
71.50
74.50
3.61
72.84
77.53
3.45
69.25
75.18
3.95
71.80
60.10
3.72
60.10
a-D-Glcp-(1!4)
Trisaccharide 2
a-L-Glcp
5.19
91.86
4.98
3.52
71.52
3.55
3.77
71.42
3.63
3.53
77.63
3.55
3.92
70.57
4.17
3.80
59.93
3.64
3.90
3.99
3.75
3.92
3.99
3.75
a-D-Glcp-(1!4)
a-D-Glcp-(1!6)
b-L-Glcp
99.46
4.92
71.36
3.53
73.08
3.72
69.4
70.65
3.70
65.43
3.62
3.38
69.55
3.53
98.07
4.61
71.59
3.22
73.10
3.58
71.85
3.54
60.50
3.82
95.76
4.98
74.55
3.55
74.52
3.63
77.48
3.55
75.20
4.17
60.04
3.64
a-D-Glcp-(1!4)
a-D-Glcp-(1!6)
99.46
4.92
98.07
71.36
3.53
71.59
73.08
3.72
73.10
69.37
3.38
69.55
70.65
3.70
71.85
65.47
3.62
60.50
a Disaccharide derived from glucoamylase-digested trisaccharide.

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G. L. Cote et al. / Carbohydrate Research 340 (2005) 257–262
261
Gel P-2 chromatography. Methylation and NMR anal-
3.3. Structural comparison of disaccharide acceptors
ysis proved the disaccharide to be a-D-glucopyranosyl-
(1!4)-L-glucose (Table 2). When this disaccharide
was tested as an acceptor for alternansucrase, TLC re-
vealed that it did indeed act as an excellent acceptor.
Qualitative comparison with maltose, the best known
acceptor for alternansucrase, showed that the disaccha-
ride, a-D-glucopyranosyl-(1!4)-L-glucose, was con-
sumed slightly more rapidly than maltose under
identical conditions and concentrations. Densitometric
analysis of the reaction mixtures after seven hours indi-
cated that the reaction mixture using a-D-glucopyr-
anosyl-(1!4)-L-glucose as the acceptor contained
approximately 10% less disaccharide acceptor, 10%
more oligosaccharide product, and an equal amount
of D-fructose, compared to the maltose acceptor reac-
tion. This was based on a single experiment, and
more extensive quantitative studies remain to be done.
However, it can be concluded that a-D-glucopyranos-
yl-(1!4)-L-glucose is at least as good an acceptor as
maltose, and possibly better.
The geometry of the active site of alternansucrase is not
known; however, maltose and a-D-glucopyranosyl-
(1!4)-L-glucose have been shown to be strong accep-
tors for alternansucrase.4 A structural comparison has
been performed on maltose and a-D-glucopyranosyl-
(1!4)-L-glucose in an attempt to gain insight into
molecular recognition of these molecules at the alternan-
sucrase active site.
Conformational preferences of b-maltose have been
previously studied,12 and the lowest energy form of b-
maltose has been identified, Figure 4A. This maltose con-
former was used for a structural comparison study in va-
cuo. a-D-Glucopyranosyl-(1!4)-L-glucose was geometry
optimized from a conformation similar to that of malt-
ose. The maltose structure, 4A, is energetically favored
over a-D-glucopyranosyl-(1!4)-L-glucose, 4B, in overall
energy, corrected energy, and free energy (see Table 3).
Glycosidic torsion angles are of considerable import-
ance in the resulting spatial arrangement of disaccharides
Figure 4. Geometry optimized structures at the B3LYP/6-311++G** level of theory for maltose, A, and a-D-Glcp-(1!4)-L-Glcp, B. Alignment of A
with B is shown below as C.

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G. L. Cote et al. / Carbohydrate Research 340 (2005) 257–262
262
Table 3. Selected energies, bond lengths, and bond angles for 4a and
4b at the B3LYP/6-11++G** level of theory
Structures 4A and 4B have their hydroxymethyl rota-
mers in the gt orientation for the nonreducible ring and
the gg orientation for the reducible ring. A notable fea-
ture of 4A is the HO-4Á Á ÁHO-3Á Á ÁHO-2Á Á ÁHO-30Á Á ÁHO-
20Á Á ÁHO-10 cooperative hydrogen-bond networkwith a
Maltose A
(Fig. 4)
a-D-Glcp-(1!4)-L-
Glcp B (Fig. 4)
Energy
DE
ZPVE
À814,727.013
À814,724.743
2.270
0
0
˚
1.960 A HO-2Á Á ÁHO-3 hydrogen bond. Interestingly,
232.064
0
232.195
2.401
the structure 4B has the HO-2 of the first glycosidic
˚
residue forming a 1.960 A hydrogen bond with the
DEcorrected
Enthalpy (H)
Entropy (S) (cal/mol K)
DG0298
246.064
155.997
0
247.602
162.004
1.139
hydroxymethyl substituent of the reducible ring. In con-
trast, structure 4B has the less extensive HO-4Á Á ÁHO-
3Á Á ÁHO-2Á Á ÁHO-60 networkand
a
HO-3 0Á Á ÁHO-
Dihedral angles
/
wH
O-6–C-6–C-5–O-5
O-60–C-60–C-50–O-50
HO-2–O-2–C-2–C-3
HO-20–O-20–C-2–0C-30
À4.68
10.67
58.97
14.43
43.06
60.49
75.69
69.10
174.09
20Á Á ÁHO-1 network. The synergistic effect of the cooper-
ative hydrogen-bond networkin structure 4A can be
seen in energy and the entropic terms, where 4A is lower
in entropy and free energy.
H
À62.03
87.36
The alignment of 4B to 4A shows remarkable similar-
ity between the two structures. Both structures have their
hydroxyl substituents in similar position through the
disaccharide, with the larg0est discrepancy being 1.009–
À176.74
Bond lengths
C-1–O-1
1.424
1.395
1.522
1.523
0.964
0.964
0.973
0.964
1.434
1.394
1.521
1.522
0.965
0.964
0.973
0.964
C-10–O-10
0
˚
C-5–C-6
1.185 A for the O-6 to O-3 comparison. The geometries
of structures 4A and 4B can reasonably achieve excellent
fit to the same binding site, apparently explaining why 4B
is such a good acceptor for alternansucrase, and why
none of this disaccharide could be isolated from acceptor
reaction mixtures that had proceeded to completion.
C-50–C-60
HO-6–O-6
HO-60–O-60
HO-2–O-2
HO-20–O-20
Hydrogen-bond lengths
HO-4Á Á ÁHO-3
2.421
2.430
1.960
2.429
2.429
HO-3Á Á ÁHO-2
HO-2Á Á ÁHO-30
HO-2Á Á ÁHO-60
HO-30Á Á ÁHO-20
HO-20Á Á ÁHO-10
References
1.960
2.411
2.570
2.408
2.559
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C-1–O1–C-40
119.33
111.38
108.86
105.76
109.24
115.82
114.13
107.27
108.40
107.21
118.04
109.60
108.91
106.11
108.89
115.69
112.30
111.38
108.39
107.28
O-1–C-1–O-5
O-10–C-10–O-50
H-1–C-1–O-5
H-10–C-10–O-50
C-1–O-5–C-5
C-10–O-50–C-50
HO-2–O-2–C-2
HO-20–O-20–C-20
HO-4–O-4–C-4
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and have been under extensive computational and
experimental investigation. The structure 4A is similar
to that reported for maltose by neutron diffraction and
X-ray crystal structures.12 The (1!4) glycosidic linkage
of 4A contains torsion angles of /H = À4.69ꢀ and
wH = 10.67ꢀ. During geometry optimization, a-D-gluco-
pyranosyl-(1!4)-L-glucose went through a transition
without barrier to the optimized structure 4B with
/ = 14.43ꢀ and w = 43.06ꢀ. This change in glycosidic
torsion angles of the 4B structure accommodated re-
markable similarity between hydrogen-bond lengths of
these disaccharides.
11. Bates, R. B.; Byrne, D. N.; Kane, V. V.; Miller, W. B.;
Taylor, S. R. Carbohydr. Res. 1990, 201, 342–345.
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