Dang Hai Dang Nguyen, Jong-Tae Park, Jae-Hoon Shim,
Phuong Lan Tran, Ershita Fitria Oktavina, Thi Lan Huong
Nguyen, Sung-Jae Lee, Cheon-Seok Park, Dan Li,
Sung-Hoon Park, David Stapleton, Jin-Sil Lee and
Kwan-Hwa Park
J. Bacteriol. 2014, 196(11):1941. DOI: 10.1128/JB.01442-13.
Published Ahead of Print 7 March 2014.
Updated information and services can be found at:
http://jb.asm.org/content/196/11/1941
These include:
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Reaction Kinetics of Substrate
Transglycosylation Catalyzed by TreX of
Sulfolobus solfataricus and Effects on
Glycogen Breakdown
Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX
of Sulfolobus solfataricus and Effects on Glycogen Breakdown
Department of Foodservice Management and Nutrition, Sangmyung University, Seoul, Republic of Koreaa; Department of Food Science and Technology, Chungnam
National University, Daejeon, Republic of Koreab; Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Koreac; Department of Biology,
University of Incheon, Incheon, Republic of Koread; Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University,
Seoul, Republic of Koreae; Graduate School of Biotechnology, Kyung Hee University, Suwon, Republic of Koreaf; Key Laboratory of Agricultural Products Processing in Jilin
Provincial Universities, Changchun University, Changchun, People’s Republic of Chinag; Department of Medicinal Chemistry and Molecular Pharmacology, Purdue
University, West Lafayette, Indiana, USAh; Department of Physiology, University of Melbourne, Victoria, Australiai
We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched
maltotetraosyl-␤-cyclodextrin (Glc4-␤-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc4-␤-CD as a glycogen mimic substrate
showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of
␣-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-␣-1,6-␤-cyclodextrin [(Glc4)2-␤-CD]; these were 61,63- and 61,64-dimaltotetraosyl-␣-1,6-␤-CD. Use of a modified
Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were
1.78 ⴛ 103 sⴚ1 and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 ⴛ 103 sⴚ1 and 0.206 mM, respectively. Also,
enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated
⬃1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting
of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action.
We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.
G
lycogen is a branched glucose polysaccharide consisting of
␣-1,4-glucosidic linkages with ␣-1,6 branches and is the major storage carbohydrate in animals equivalent to starch in plants
(1, 2). Glycogen constitutes more than half of the Escherichia coli
cell mass when the bacteria are grown under conditions of nitrogen shortage but with an excess carbon source (3–6). Glycogen
accumulation commences during the lag phase, is maximal during the stationary phase (⬃15 h), and decreases over the next
several hours in many bacteria, such as Bacillus subtilis (7, 8).
Glycogen biosynthesis in bacteria and archaea is accomplished by
production of ADP-glucose and formation of ␣-1,4 glucosidic
chains and ␣-1,6-linked glucan branches (9), whereas UDP-glucose initiates glycogen biosynthesis in mammalian muscle and
liver. Thus, the enzyme is involved in glycogen metabolism in
bacteria, and archaea share significant homology and enzymatic
mechanisms (9). In contrast, breakdown of glycogen is initiated
by debranching enzymes catalyzing hydrolysis of the ␣-1,6-glucosidic linkages of glycogen, amylopectin, and pullulan. Such enzymes are widely distributed in nature, being found in animals,
bacteria (10–14), archaea (15–17), and plants. The glycogen degradation process in animals and yeasts is well known, in which the
debranching enzymes exhibit two distinct activities—a glucanotransferase action and amylo-1,6-glucosidase activity (2, 18, 19).
However, the glycogen breakdown process is unknown or the
mode of action of debranching enzymes is not well understood in
bacteria and archaea.
One of the main reasons that debranching enzymes of micro-
June 2014 Volume 196 Number 11
organisms have been poorly studied is the difficulty in identifying
intermediate reaction products because the substrate (glycogen)
has a complex structure, making it difficult to describe the reaction mechanism in detail. Thus, we prepared maltodextrinbranched cyclodextrin as a glycogen mimic to study branch chain
specificity of the debranching enzyme. TreX, a glycogen debranching enzyme cloned from the trehalose biosynthesis gene
cluster of Sulfolobus solfataricus P2, catalyzes the debranching reaction of the branch chain of glycogen into maltodextrin. It belongs to the GH13 (glycoside hydrolase 13 in the CAZy classification) family and shows high sequence similarity to isoamylase
(20). However, our previous study showed that TreX exhibits both
hydrolysis and transglycosylation activities by catalyzing the
breakage and transfer of ␣-1,4-glucan oligosaccharides among
chains (20). We also obtained a three-dimensional structure of
TreX and identified two distinct active site configurations, in line
with the bifunctional nature of the enzyme (21). The bifunctional
debranching enzymes from mammalian tissues transfer the maltotetraosyl moiety to the nonreducing end of glucose, elongating
Received 12 December 2013 Accepted 3 March 2014
Published ahead of print 7 March 2014
Address correspondence to Kwan-Hwa Park, [email protected].
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JB.01442-13
Journal of Bacteriology
p. 1941–1949
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1941
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Dang Hai Dang Nguyen,a Jong-Tae Park,b Jae-Hoon Shim,c Phuong Lan Tran,a Ershita Fitria Oktavina,d Thi Lan Huong Nguyen,a
Sung-Jae Lee,e Cheon-Seok Park,f Dan Li,g Sung-Hoon Park,h David Stapleton,i Jin-Sil Lee,a Kwan-Hwa Parka,e
Nguyen et al.
MATERIALS AND METHODS
Materials. Thin-layer chromatography (TLC) plates were purchased
from Whatman International, Ltd. (Maidstone, Kent, United Kingdom),
Ni-nitrilotriacetic acid (NTA) Superflow matrices were obtained from
Qiagen (Hilden, Germany), and Bacto tryptone and yeast extract were
from Difco (Detroit, MI). Other reagents were purchased from Sigma
Chemical Co. (St. Louis, MO), Merck (Darmstadt, Germany), Junsei
Chemical Co. (Tokyo, Japan), Showa Chemicals, Inc. (Tokyo, Japan), and
Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Purification of TreX. The glycogen debranching enzyme (GDE) of S.
solfataricus P2 (TreX) was expressed in E. coli MC1061 [F⫺ araD139
recA13 ⌬(araABC-leu)7696 galU galK ⌬lacX74 rpsL thi hsdR2 mcrB].
Transformants were cultured in Luria-Bertani (LB) medium (1% [wt/vol]
Bacto tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) containing ampicillin (100 ␮g ml⫺1) at 37°C. TreX tagged with six His residues
was purified on a 1- by 4-cm Ni-NTA column (Ni-NTA Superflow; Qiagen) as described previously (22), and enzyme purity was confirmed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
TreX assays. TreX activities were measured as described previously
(23). Hydrolytic activity was assayed at 75°C in 50 mM sodium acetate
buffer (pH 5.5) using 0.5% (wt/vol) amylopectin as the substrate. The
reducing power of hydrolysis products was determined using the dinitrosalicylic acid method (24). One unit of TreX was defined as the amount of
enzyme releasing 1 ␮mol equivalent of maltose min⫺1. Protein concentrations were determined using bovine serum albumin (BSA; Sigma
Chemical Co.) as a standard (25).
Synthesis of maltodextrin-branched cyclodextrin and branched
maltoheptaose. Five milliliters of a solution of concentrated maltosyl
transferase from Thermatoga maritima (TmMT) was added to a solution
of 5% (wt/vol) maltosyl-␤-cyclodextrin (Glc2-␤-CD) and 10% (wt/vol)
soluble starch in McIlvaine’s buffer (pH 6.5), and the reaction proceeded
at 70°C for 3 days. The solution was next heated to 100°C for 20 min to
inactivate the enzyme and loaded onto a C18 Sep-Pak column to remove
small oligosaccharides. The substrate was purified by several-stage preparative high-performance liquid chromatography (HPLC). Dimaltotetraosyl-␤-cyclodextrin [(Glc4)2-␤-CD] was prepared by a modification of
the method of Kang et al. (26). To prepare maltotetraosyl-␣-1,6 maltoheptaose, Glc4-␤-CD was hydrolyzed with Pyrococcus furiosus amylase
(PfTA), and the product was purified on a butyl-Sepharose column (27).
Cloning, expression, and purification of yeast glycogen debranching enzymes. To clone the GDE of Saccharomyces cerevisiae, four PCR
primers were used: forward1 (5=-CGG GCC ATG GGA ATG AAT AGA
TCA TTA CTG CTA CGT TTG TCG-3= [NcoI restriction site underlined]) and reverse1 (TGT CAT AGA TAA GCT TTG TAC AAA CAA
TGT AGA [HindIII restriction site underlined]) amplifying bases 1 to
3123 (the large fragment) and forward2 (TCT ACA TTG TTT GTA CAA
AGC TTA TCT ATG ACA [HindIII restriction site underlined]) and reverse2 (CGC GAA GCT TTC AGG AAT CAT CTT CGT AGG CAT CCC
ATA A [HindIII restriction site underlined]) amplifying bases 3124 to
4608 (the small fragment). These primers were designed with reference to
the full genome sequence in GenBank. The large fragment was PCR amplified from genomic DNA of S. cerevisiae S288c. The PCR product was
digested with NcoI and HindIII and ligated with identically cut pPRoEX
HTa (Invitrogen, Carlsbad, CA). The recombinant plasmid was used as a
vector for subsequent cloning of the small fragment, which was amplified
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using the forward2 and reverse2 primers followed by HindIII digestion.
Finally, the small and large fragments were ligated, and the recombinant
gene was sequenced (in pPRoEX Hta). The final recombinant plasmid was
designated pPRoEXGDB1.
To overexpress GDB1, pPRoEXGDB1 was transformed into E. coli
BL21(DE3). A transformant was cultured in 1 liter LB medium (10 g
tryptone, 5 g yeast extract, 5 g sodium chloride) at 37°C with shaking (250
rpm), and isopropyl-␤-D-thiogalactopyranoside (IPTG) was added to
0.01 mM at an optical density at 600 nm (OD600) of 1.0. After induction,
the culture was cooled to 25°C and incubated for 24 h. Cells were harvested by centrifugation (8,000 ⫻ g, 20 min), and the pellet was sonicated
(VC-600; Sonics & Materials, Danbury, CT) in an ice-cold water bath.
After removal of cell debris, GDB1 was purified by Ni2⫹-nitrilotriacetate
(Ni-NTA) column chromatography (Qiagen) following the manufacturer’s method.
Overexpression and purification of GlgP. The glycogen phosphorylase gene (glgP) was amplified by PCR from genomic DNA of E. coli K-12,
with the addition of restriction sites (underlined below) required for cloning. The primers were GlgP/XbaI (5=-CGG AAA AAT CTA GAA CCC
TTT GGC CCC GTT C-3=) and GlgP/NdeI (5=-GAA ACG CCC ATA TGA
ATG CTC CGT TTA CAT ATT C-3=). The PCR product was digested with
NdeI and XbaI and ligated into identically cut pTKNd6⫻H. The resulting
construct (pTKNd6⫻H_GlgP) was transformed into E. coli MC1061, and
transformants were cultured on LB medium with kanamycin (50 ␮g
ml⫺1). GlgP was purified by Ni-NTA chromatography, and enzyme activity on glycogen as the substrate was measured using high-performance
anion-exchange chromatography (HPAEC).
Isolation of glycogen from E. coli. E. coli MC4100 [araD139 deoC1
flbB5301 ptsF25 rbsR relA1 rpsL150 ⌬(argF-lac)U169] was cultured in M63
minimal salts (1 liter) supplemented with 0.5% (wt/vol) maltose as a
carbon source at 37°C for 20 h with shaking (250 rpm). Cells were harvested by centrifugation (6,000 ⫻ g) at 4°C for 20 min, washed with
ice-cold water, and resuspended in 5 ml 50 mM sodium acetate (pH 4.5).
Glycogen was isolated from this suspension and quantified as described by
Park et al. (28).
Reaction of yeast debranching enzyme with branched Glc4␤CD.
The purified yeast debranching enzyme (1.5 mU) was added to 1% (wt/
vol) solutions of Glc4-␤-CD or Glc1-␤-CD in 50 mM NaOAc buffer (pH
6.0). Reactions proceeded for 0.5 and 1 h at 37°C, and products were
analyzed using TLC and HPAEC.
Preparation of phosphorylase-limited dextrin from glycogen. Phosphorylase-limited dextrin was prepared as described by Hers et al. (29).
Glycogen from E. coli (500 mg) was incubated with 37.5 ␮g phosphorylase
in 20 ml 0.1 M sodium phosphate buffer (pH 6.5) in dialysis tubing and
dialyzed against the same buffer. After 24 h, 20 ␮g phosphorylase a was
added to the contents of the bag, and the incubation was continued for an
additional 24 h. The reaction was terminated by heating at 100°C for 5
min, and insoluble material was removed by centrifugation. The resulting
solution was dialyzed against water and freeze-dried.
Reaction of TreX with branched Glc4-␤-CD. TreX (1.5 mU) was incubated with Glc4-␤-CD (0.5 mM) in 50 mM sodium acetate buffer (pH
5.5) in the presence of 10% (vol/vol) dimethyl sulfoxide (DMSO) at 70°C.
Samples were taken at 1, 5, 10, 30, 60, and 90 min and heated at 100°C for
10 min. The samples were centrifuged and analyzed by TLC and HPAEC.
Reaction of glycogen phosphorylase with glycogen and phosphorylase-limited dextrin from glycogen, in the presence of TreX. Glycogen
phosphorylase was incubated with 1% (wt/vol) glycogen or phosphorylase-limited dextrin in the presence (0.2 to 1 mU) or absence of TreX in 50
mM sodium phosphate buffer (pH 6.5) at 37°C. Reactions proceeded for
15, 30, and 60 min, and samples were heated at 100°C for 5 min. Insoluble
material was removed by centrifugation, and the resulting solutions were
analyzed by HPAEC. Glc-1P production was quantified using a standard
curve constructed with the aid of authentic material.
Analysis of reaction products. (i) TLC analysis. Silica gel K5F TLC
plates (Whatman) were activated for 1 h at 110°C. Samples were spotted
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the branch chain with an ␣-1,4-glycosidic linkage that is immediately hydrolyzed by glycogen phosphorylase (GlgP). However, unlike the animal debranching enzyme, TreX transfers the glucan
moiety by forming an ␣-1,6 glycosidic linkage, resulting in translocation of the branch point within the molecule (20). The breakdown processes including the debranching enzyme are likely to
have a different mechanism. Therefore, this study aimed to elucidate the mechanism of substrate transglycosylation of the TreX
enzyme and its effect on the glycogen breakdown process.
Substrate Transglycosylation Catalyzed by TreX
FIG 1 Thin-layer chromatography analysis of products from reaction mixtures containing yeast glycogen debranching enzyme. Product numbers: 1,
glucose; 2, ␤-CD; 3, glucose-␣-1,6-␤-CD; 4, maltotriosyl-␣-1,6-␤-CD; 5,
maltotetraosyl-␣-1,6-␤-CD; 6, maltoheptaosyl-␣-1,6-␤-CD; 7, maltodecaosyl-␣-1,6-␤-CD; 8, maltotridecaosyl-␣-1,6-␤-CD; 9, maltotetradecaosyl-␣-1,6-␤-CD.
onto plates using a pipette, and the plates were placed in a TLC chamber
containing n-butanol– ethanol–water at 5:5:3 (vol/vol/vol). Chromatography was conducted at room temperature. Reducing sugars were detected using the naphthol-sulfuric acid (H2SO4) method (30). Each plate
was thoroughly dried and developed by rapid dipping into a methanol
solution containing 3 g N-(1-naphthyl)-ethylene-diamine and 50 ml concentrated H2SO4 per liter. Each plate was dried and placed in an oven at
110°C for 10 min; purple-black spots appeared on a white background.
(ii) HPAEC. The reaction mixtures incubated with TreX were boiled
for 10 min, centrifuged at 12,000 ⫻ g for 10 min, and filtered using a
membrane filter kit (pore diameter, 0.45 ␮m) (Millipore, Billerica, MA).
Products were applied to a 0.4- by 25-cm CarboPac PA-1 column (Dionex, Sunnyvale, CA) and analyzed on an HPAEC platform fitted with an
ED40 pulsed amperometric detector (Dionex). Sixty-microliter aliquots
of sample were injected. Elution was achieved using a NaOAc gradient in
150 mM NaOH (increasing from 60 to 180 mM NaOAc over 0 to 10 min,
from 180 to 240 mM over 10 to 16 min, from 240 to 300 mM over 16 to 27
min, from 300 to 360 mM over 27 to 44 min, and from 360 to 372 mM
over 44 to 55 min). The flow rate was 1 ml min⫺1.
(iii) Isolation and identification of transfer products. TreX (3 mU
mg⫺1 of substrate) was reacted with 0.5% (wt/vol) Glc4-␤-CD in 50 mM
sodium acetate buffer (pH 5.5) at 70°C for 5 min. The reaction mixture
was next heated at 100°C for 10 min and passed through a 0.45-␮m-pore
Millex-HV filter (Millipore) prior to two-step HPLC (Jaigel W-251 col-
June 2014 Volume 196 Number 11
umn, 2 ⫻ 50 cm; W-252 column, 2 ⫻ 50 cm) (Japan Analytical Industry,
Tokyo, Japan) using distilled water to elute bound materials (flow rate, 3.5
ml min⫺1). Fractions were analyzed by HPAEC. To identify positional
isomers of transfer products, (Glc4)2-␤-CD was converted into dimaltosyl-␤-cyclodextrin [(Glc2)2-␤-CD] by reaction with ␤-amylase (30 U) at
37°C for 12 h, followed by heating at 100°C for 5 min and filtration
through a 0.45-␮m-pore diameter membrane to improve isomer separation by HPLC. An 1100 HPLC system (Agilent Technologies, Inc., Waldbronn, Germany) fitted with a Hypercarb column (150- by 4.6-mm internal diameter) (Thermo-Scientific, Loughborough, United Kingdom) and
an evaporative light scattering detector (ELSD 2000; Alltech Associates,
Inc., Deerfield, IL) was used in the analysis. Elution was performed at 60°C
using an isocratic solvent system of acetonitrile and water (20:80 [vol/
vol]) at a flow rate of 1 ml min⫺1.
(iv) Mathematical characterization of the TreX reaction with Glc4␤-CD. The kinetics of substrate transglycosylation and hydrolysis were
calculated using the minimal kinetic scheme for substrate transglycosylation proposed previously by Kawai et al. (31) and Gusakov et al. (32).
Substrate transglycosylation by TreX was considered to proceed as
shown in equations 1 and 2 below.
k6
(Glc4)2-␤-CD → Glc4-␤-CD ⫹ Glc4
(2)
Equation 1 yields the rate equations
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FIG 2 Thin-layer chromatography analysis of the reaction products yielded
by TreX acting on 0.5 mM (A) and 0.05 mM (B) Glc4-␤-CD. Glc1 to Glc7,
standard maltooligosaccharides from glucose to maltoheptaose; ␤-CD, ␤-cyclodextrin; Glc4-␤-CD, 6-O-␣-maltotetraosyl-␤-cyclodextrin; (Glc4)2-␤-CD,
dimaltotetraosyl-␤-cyclodextrin.
Nguyen et al.
3, mixture of isomers of dimaltotetraosyl-1,6-␤-CD; 4, maltotetraose.
v0
kcat[S] ⫹ kcat2[S]2
⫽
[E]t
[S]2
Km ⫹ [S] ⫹
Km2
V␤-CD ⫽
2kcatKm2关S兴 ⫹ kcat2关S兴2
KmKm2 ⫹ Km2关S兴 ⫹ 关S兴2
(3)
(4)
where Km and kcat are parameters for the hydrolysis reaction, Km2 and kcat2
are the parameters for the transglycosylation reaction, and E and S represent the enzyme and the substrate, respectively.
To determine the transglycosylation kinetic parameters, the hydrolytic
parameters Km and kcat were measured in reactions with Glc4-␤-CD as the
substrate at 0.05 to 0.3 mM. The transglycosylation kinetic parameters
were determined at higher substrate concentrations (0.5 to 2 mM). Additionally, the first-order reaction rate for hydrolysis of (Glc4)2-␤-CD was
separately determined after isolation of (Glc4)2-␤-CD.
RESULTS
The glycogen mimic substrate (Glc4-␤-CD) is suitable for characterization of the glycogen breakdown process. To validate the
suitability of the glycogen mimic substrate for a glycogen study,
Glc4-␤-CD was tested with a mouse liver extract and yeast debranching enzymes whose reaction modes are well known. TLC
(Fig. 1) and HPAEC (data not shown) yielded information on the
reaction products of mouse liver and yeast debranching enzymes
acting on Glc4-␤-CD (Fig. 1). When the glycogen debranching
enzyme of yeast (GDE) was incubated with Glc4-␤-CD, Glc1␤-CD (product no. 3 in Fig. 1A) and a series of transfer products—Glc7-␤-CD to Glc2-␤-CD (no. 6, 7, 8, and 9 in Fig. 1A)—
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were obtained. The maltotriosyl moiety of Glc4-␤-CD was
transferred to another molecule of Glc4-␤-CD and also to other
materials (Fig. 1). Thus, the GDE of yeast elongated branch chain
length via the formation of ␣-1,4-glucosidic linkages in the absence of glycogen phosphorylase. One product, Glc1-␤-CD (no.
3), was further hydrolyzed by the yeast GDE to glucose (product 1)
and ␤-CD (product 2). In addition, Glc3-␤-CD (product 4) was
produced by GDE action; glucose was released from Glc4-␤-CD.
However, no (Glc4)2-␤-CD was produced when yeast GDE reacted with Glc4-␤-CD. HPAEC and analysis of Rf values by TLC
also identified Glc7-␤-CD and Glc1-␤-CD. Formation of Glc7␤-CD was confirmed by incubating the putative product with
isoamylase; Glc7 and ␤-CD were produced. These results show
that glycogen degradation by the yeast debranching enzyme can be
followed with Glc4-␤-CD as a substrate analog.
TreX has both hydrolytic and transglycosylation activity at
high substrate concentrations (>0.5 mM). The enzyme was incubated with Glc4-␤-CD (0.5 mM) in 50 mM sodium acetate buffer (pH 5.5) in the presence of 10% (vol/vol) DMSO at 70°C.
Samples were taken at various time intervals and heated at 100°C
for 10 min prior to analysis by TLC and HPAEC. Unlike what was
noted when the debranching enzymes of yeast and mouse liver
extract were studied, TreX did not generate ␣-1,4 hydrolysis products such as Glc3-, Glc2-, or Glc1-␤-CD. TreX reacted with Glc4␤-CD to initially form the transglycosylation product (Glc4)2-␤CD. These concentrations then decreased gradually, whereas
␤-CD and maltotetraose concentrations increased, as the reaction
time was extended (Fig. 2, 3, and 4). When the Glc4-␤-CD con-
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FIG 3 Time course HPAEC analysis of the reaction products of TreX acting on 0.5 mM (A) and 0.05 mM (B) Glc4-␤-CD. 1, maltotetraosyl-1,6-␤-CD; 2, ␤-CD;
Substrate Transglycosylation Catalyzed by TreX
treatment (A) and after ␤-amylase treatment (B). The positional isomers of
(B) were analyzed by HPLC using a Hypercarb column (C). Products: 1, a
mixture of 61,63- and 61,64-dimaltotetraosyl-␤-CD; 2, a mixture of 61,63and 61,64-dimaltosyl-␤-CD; 2A, 61,63-dimaltosyl-␤-CD; 2B, 61,64-dimaltosyl-␤-CD.
FIG 4 Time course analysis of TreX products produced when the enzyme
acted on 0.5 mM (A) and 0.05 mM (B) Glc4-␤-CD. , maltotetraose; ●, ␤-CD;
, maltotetraosyl-1,6-␤-CD; 䊐, dimaltotetraosyl-␤-CD.
centration was 0.05 mM, the Glc4-␤-CD level decreased gradually,
whereas the ␤-CD and maltotetraose concentrations increased
with the reaction time (Fig. 2B to 4B). As the positional isomers of
the (Glc4)2-␤-CD transfer products were not separated on the
Hypercarb column, they were first converted to (Glc2)2-␤-CD by
␤-amylase (Fig. 5A and B) and analyzed by HPLC using the Hypercarb column. As shown in Fig. 5C, comparison of the chromatographic profile with that of (Glc2)2-␤-CD reference materials
(21) indicated that the transfer product was a mixture of 61,63and 61,64-(Glc4)2-␤-CD at a molar ratio close to unity. Additionally, the formation of 61,64-(Glc4)2-␤-CD observed in the present
study and the synthesis of 61,64-(Glc2)2-␤-CD reported by Kang et
al. (26) support the idea that the transfer products produced from
branch chains varying widely in size are similar.
Kinetics of hydrolysis and transglycosylation of Glc4-␤-CD
by TreX. The enzyme was added to Glc4-␤-CD solutions of various concentrations (0.05 to 2 mM). As transglycosylation was observed at substrate concentrations of ⬎0.5 mM (Fig. 2 and 3), a
kinetic analysis was conducted using two ranges of substrate con-
June 2014 Volume 196 Number 11
centration. The Km and kcat of hydrolysis were assessed at substrate
concentrations of 0.05 to 0.3 mM, and the Km2 and kcat2 of transglycosylation were determined at 0.5 to 2 mM. Data plotting
yielded the initial velocities of Glc4 production at various substrate
concentrations. A Lineweaver-Burk plot (data not shown) showed
that the Km and kcat of hydrolysis were 0.206 ⫾ 0.003 mM and
(2.57 ⫾ 0.334) ⫻ 103 s⫺1, respectively, and the kcat/Km ratio was
(12.42 ⫾ 0.162) ⫻ 103 mM⫺1· s⫺1. To determine the kinetic parameters of transglycosylation, TreX was reacted with Glc4-␤-CD
at 0.5 to 2 mM. The formation of ␤-CD was quantified by HPAEC
to obtain initial reaction velocities. Equation 4 was used to estimate the transglycosylation parameters Km2 ⫽ 3.39 ⫾ 0.057 mM,
kcat2 ⫽ (1.78 ⫾ 0.03) ⫻ 103 s⫺1, and kcat2/Km2 ⫽ (0.54 ⫾ 0.01) ⫻
103 mM⫺1·s⫺1, which indicated that transglycosylation occurred
principally at high substrate concentrations. The branched glycosyl chain of the Glc4 substrate was transferred to a hydroxyl group
of an acceptor molecule other than water, and the extent of hydrolysis
was reduced. Thus, transglycosylation may influence the overall reaction rate. As the reaction proceeds, hydrolysis began to dominate
over transglycosylation, leading to the production of Glc4 and ␤-CD
from the TreX transglycosylation products (Fig. 4).
TreX has high specific activity for a wide range of branch
chain lengths. Glycogen contains branch chains of various
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FIG 5 HPAEC analysis (A and B) of transfer products prior to ␤-amylase
Nguyen et al.
O-␣-maltosyl-␤-cyclodextrin), Glc4-␤-CD (6-O-␣-maltotetraosyl-␤-cyclodextrin), Glc6-␤-CD (6-O-␣-maltohexaosyl-␤-cyclodextrin), and Glc8-␤-CD
(6-O-␣-maltooctaosyl-␤-cyclodextrin). (A) TLC analysis of the substrate concentration at various reaction times; (B) values of the kcat/Km ratio.
lengths. To explore TreX activities on various branch chains, Glc2to Glc10-␤-CDs were incubated with TreX, and the products were
analyzed by HPAEC and TLC. Unexpectedly, TreX was active on
all branch chains studied and the kcat/Km ratio increased as the
branch chain degree of polymerization (DP) rose (Fig. 6).
Action of TreX on branched maltotetraosyl-␣-1,6-maltoheptaose. Unlike cyclodextrin, glycogen is a linear glucan. To confirm that TreX was active on linear glucans, the enzyme was incubated with maltotetraosyl-␣1,6-maltoheptaose, yielding three
major products after a 10-min reaction. The substrate was hydrolyzed to maltoheptaose and maltotetraose. In parallel, the maltotetraose moiety was transferred to another substrate molecule,
producing dimaltotetraosyl maltoheptaose, which was in turn
DISCUSSION
Branched cyclodextrins as substrate mimics were used previously
to characterize yeast debranching enzymes (33). However, transfer products were only indirectly identified by further hydrolysis
with pullulanase, in which dibranched products cannot be detected using such a method. Kang et al. (26) synthesized di-O-␣maltosyl-␤-cyclodextrin [(Glc2)2-␤-CD] from 6-O-␣-maltosyl␤-cyclodextrin via transglycosylation catalyzed by TreX. This
finding suggested that TreX transfers the maltosyl residue of Glc2␣-1,6-␤-CD with formation of an ␣-1,6-glycosidic linkage. TreX
also yielded (Glc4)2-␤-CD containing an ␣-1,6-glucosidic linkage
(Fig. 7). Similarly, 63-maltotetraosyl maltoheptaose, similar to a
glycogen branch, produced the transfer product of (Glc4)2-maltoheptaose (data not shown). Thus, transglycosylation occurs via
formation of an ␣-1,6-glycosidic linkage when either branched
linear maltodextrin or Glc4-␤-CD serves as the substrate (data not
TABLE 1 Effect of TreX on formation of glucose-1-phosphatea
Substrate
Limited dextrin, 10 mg/ml
Glycogen
1 mM
0.5 mM
a
TreX
concn (mU)
Glucose-1-phosphate concn (mM) at:
15 min
30 min
60 min
Rate constant (mmol
liter⫺1·min⫺1) ⫻ 10⫺3
0.0
0.2
0.5
0.030 ⫾ 0.002
0.030 ⫾ 0.002
0.028 ⫾ 0.002
0.052 ⫾ 0.004
0.049 ⫾ 0.003
0.048 ⫾ 0.003
0.071 ⫾ 0.058
0.067 ⫾ 0.042
0.064 ⫾ 0.040
1.15 ⫾ 0.09
1.08 ⫾ 0.07
1.04 ⫾ 0.06
0.0
0.2
0.5
0.086 ⫾ 0.006
0.092 ⫾ 0.003
0.092 ⫾ 0.001
0.137 ⫾ 0.011
0.180 ⫾ 0.012
0.171 ⫾ 0.002
0.242 ⫾ 0.019
0.331 ⫾ 0.022
0.323 ⫾ 0.005
3.91 ⫾ 0.31
5.48 ⫾ 0.36
5.71 ⫾ 0.39
0.0
0.2
0.5
0.045 ⫾ 0.004
0.045 ⫾ 0.004
0.045 ⫾ 0.012
0.062 ⫾ 0.05
0.078 ⫾ 0.08
0.075 ⫾ 0.20
0.132 ⫾ 0.012
0.148 ⫾ 0.016
0.145 ⫾ 0.039
2.13 ⫾ 0.19
2.44 ⫾ 0.13
2.38 ⫾ 0.16
Glycogen or phosphorylase-limited dextrin was incubated with TreX (0.2 to 0.5 mU) in the presence of 60 mU of glycogen phosphorylase at 37°C for 15 to 60 min.
1946
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FIG 6 TreX activities toward various branched cyclodextrins: Glc2-␤-CD (6-
further hydrolyzed to maltotetraose and maltoheptaose (data not
shown). This supports the suggestion that TreX exerts ␣-1,6transfer activity on branched glucans. The reaction did not yield
either maltooctaose (Glc8) or maltoundecaose (Glc11), indicating
that the maltotetraosyl moiety was not transferred via formation
of an ␣-1,4 linkage but rather an ␣-1,6 linkage.
Transglycosylation enhances the rate of glycogen dephosphorylation. To explore the effect of transglycosylation on glycogen degradation in a model system, glycogen (E. coli MC4100) was
incubated with glycogen phosphorylase in the presence or absence
of TreX. The latter enzyme significantly increased the rate of
Glc-1P formation (5.48 ⫻ 10⫺3 mmol·liter⫺1·min⫺1 compared to
the control value of 3.91 ⫻ 10⫺3 mmol·liter⫺1·min⫺1). Glc-1P was
formed more rapidly when glycogen was present at 1.0 mM than
that at 0.5 mM (Table 1), which was in line with the observation
that transglycosylation occurred preferentially at higher concentrations of Glc4-␤-CD (Fig. 2 and 3). In contrast, TreX activity on
limited dextrin did not vary with substrate concentration (Table
1). A slight increase in the rate in the absence of TreX was likely
due to the incomplete limited dextrin preparation. These results
indicate that shifting branch chains to the outer layers of glycogen
was achieved via transglycosylation, thus, increasing the number
of nonreducing glucose moieties available to phosphorylase
(Table 1).
Substrate Transglycosylation Catalyzed by TreX
shown). Additionally, transglycosylation of branch chains can occur between two molecules or within a molecule. Therefore, glycogen consisting of a large number of branch chains in a molecule
possibly includes a transglycosylation step in which the same glycogen molecule has both acceptor and donor sites. The branch
chains occupy ⬃62% of the space within a glycogen molecule,
indicating an extremely high density between branch chains in
glycogen (34). Therefore, we assume that the local concentrations
of donors and acceptors within an individual glycogen molecule
may be sufficiently high to trigger transglycosylation. This observation indicates that TreX exerts transfer activity, forming ␣-1,6glycosidic linkages in the outer layers of glycogen. In addition, the
extent of transglycosylation is probably much higher when authentic glycogen is the substrate, rather than artificial branched
␤-CDs. Tabata et al. observed that the transferase activity of yeast
debranching enzyme acting on limited dextrin was higher than
that when a substrate mimic, Glc4-␤-CD, was employed (33).
The substrate specificity to various branch chain lengths of
glycogen was determined in detail to determine which branch
chain could be efficiently employed during glycogen breakdown.
Possible transglycosylation reactions of microbial debranching
enzymes require more intensive study, as the enzymes of archaea
and bacteria have received little attention compared to those of
animals and yeasts. A previous study indicated that TreX accepts a
wide spectrum of substrates, the chains of which have DP values of
3 to 6 (23). In the present study, we further analyzed the effects of
branch chain length using a wide range of substrates. Unexpectedly, TreX was active on branched chains of DP2 to DP10; transfer
products were evident with all tested substrates. This result agrees
with the suggestion of Woo et al. (21) that subunit tetramerization
generates a channel-like cavity that allows long branch chains to
be accommodated. An isoamylase from Pseudomonas spp. had the
highest specific activity toward a branch chain of DP7, but debranching of the DP2 chain was minimal (data not shown). The
debranching enzyme from Nostoc punctiforme exhibits a catalytic
June 2014 Volume 196 Number 11
preference for longer maltooligosaccharides (DP, ⬎8) (11). In
contrast, GlgX, a debranching enzyme from E. coli, is active mainly
against DP4 chains (5, 10), and AmyX from B. subtilis is specific
for DP3 to -6 chains (7). A substrate with a branch chain length of
DP4 can be considered a suitable substrate for kinetic analysis
because such a substrate is within the range of preferable chain
length for TreX. Moreover, a limited dextrin containing a branch
chain of DP4 is produced via the action of glycogen phosphorylase
and is also conveniently studied in relation to mammalian debranching enzyme. The average branch chain lengths of glycogen in
E. coli (28), S. solfataricus (35), and B. subtilis (36) are DP7 to -11,
and the structures are expected to be similar to that of mammalian
glycogen (DP13). Meléndez-Hevia et al. (34) and Meléndez et al.
(37) developed a mathematical model of the mammalian glycogen
molecule. An optimized glycogen structure features chains that
are an average of 13 glucose residues in length. The chains are
exposed to the action of phosphorylase because they are sufficiently flexible to allow binding to the enzyme active site. Moreover, the presence of a large number of such chains ensures that a
phosphorylase active site rendered vacant by loss of a chain can be
immediately filled by another adjacent chain (34).
Branch chains on the glycogen surface are accessible by phosphorylase. The three-dimensional structure of mammalian glycogen phosphorylase features a narrow 30-Å crevice that binds glycogen, accommodating 4 to 5 glucosyl residues (38, 39). Similarly,
archaeal phosphorylase acting on external chains releases glucose,
thus allowing the enzyme to act on internal chains on the same
surface. A consequence of transglycosylation of branch chains is
an increase in chain density on the outer glycogen layer. Internal
chains are shifted outward, increasing the number of nonreducing
ends available to phosphorylase (Fig. 8). The kinetic data (Table 1)
revealed that such outward transfer provides many points of attack for phosphorylase, increasing glucose release. As shown in
Fig. 2A, transglycosylation occurs preferentially at high substrate
concentrations (⬃0.5 mM Glc4-␤-CD). Branch chains occupy
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FIG 7 Schematic diagram of hydrolysis and transglycosylation of maltotetraosyl ␤-cyclodextrin (Glc4-␤-CD) catalyzed by TreX.
Nguyen et al.
⬃62% of the space within a glycogen molecule, indicating an extremely high density between branch chains in glycogen (34).
Therefore, we can assume that the local concentrations of branch
chains in the outer tier of the glycogen molecule may be sufficiently high to enable transglycosylation.
Glycogen metabolism is highly interconnected with a wide variety of cellular processes, including stringent response, Mg2⫹
availability, and osmotic pressure (40). Therefore, glycogen as a
reserve macromolecule has little effect on the internal osmotic
pressure of the cell (2). Maintaining osmotic pressure during
the glycogen degradation is important. Instead of hydrolysis of the
branch chain, in this model, transglycosylation may maintain the
level of osmotic pressure and fluid viscosity, as the release of linear
branch chains (DPs of ⬎5 to 12) by hydrolysis may cause a drastic
increase in osmotic pressure and viscosity of cellular fluid,
whereas Glc-1P from the transferred branch chains can be immediately metabolized into the glycolytic pathway without a significant change in osmotic pressure. A mutant enzyme that has high
hydrolysis activity with low transglycosylation activity or cloning
of a novel debranching enzyme from another microbial source
having low transglycosylation selectivity is required for further
study. Moreover, transglycosylation inhibits hydrolysis (31). Depending on the environment of a given microorganism, debranching (hydrolysis) of glycogen may be controlled by product
inhibition and may be further hindered by transglycosylation side
reactions. Until recently, the physiological function of the microbial debranching enzyme has been considered to be principally
hydrolysis. However, we suggest that transglycosylation may also
be involved in glycogen degradation. Similarly, trehalose biosynthesis in Sulfolobus may proceed via transglycosylation catalyzed
by TreX in association with the actions of enzymes encoded by
treZY (15, 16), and glucan chains shifted to the outer layer by TreX
may facilitate the actions of maltooligosyl trehalose synthase
(TreY) and maltooligosyl trehalose hydrolase (TreZ). We have
shown that just as mammalian debranching enzymes exert dual
functions, transglycosylation in archaea may assist in glycogen
breakdown by forming and shifting ␣-1,6-glucosidic linkages by
chain transglycosylation to the outer layers of glycogen. However,
in the present study, we used kinetic data to explore the possible
contribution of transglycosylation to glycogen breakdown in a
model system in which TreX from S. solfataricus was introduced to
glycogen and glycogen phosphorylase from E. coli. Therefore, a
1948
jb.asm.org
full set of enzymes involved in the glycogen metabolism of a strain
of bacteria or archaea should be introduced, and their actions
should be analyzed in concert.
ACKNOWLEDGMENTS
This study was supported by the Basic Research Program of the National
Research Foundation (grant no. 2012R1A1A2005012) and the Next Generation BioGreen21 Program (SSAC, grant no. PJ009086), Rural Development Administration, Republic of Korea.
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