Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreXof Sulfolobus solfataricus and Effects on Glycogen Breakdown

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

Department of Foodservice Management and Nutrition, Sangmyung University, Seoul, Republic of Koreaa; Department of Food Science and Technology, ChungnamNational 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 JilinProvincial Universities, Changchun University, Changchun, People’s Republic of Chinag; Department of Medicinal Chemistry and Molecular Pharmacology, PurdueUniversity, 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, branchedmaltotetraosyl-�-cyclodextrin (Glc4-�-CD), and natural glycogen to better understand substrate transglycosylation and the ef-fect thereof on glycogen debranching in microorganisms. The validation test of Glc4-�-CD as a glycogen mimic substrateshowed 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 malto-tetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimal-totetraosyl-�-1,6-�-cyclodextrin [(Glc4)2-�-CD]; these were 61,63- and 61,64-dimaltotetraosyl-�-1,6-�-CD. Use of a modifiedMichaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were1.78 � 103 s�1 and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 � 103 s�1 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 reac-tion 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 shiftingof 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 deb-ranching enzyme.

Glycogen is a branched glucose polysaccharide consisting of�-1,4-glucosidic linkages with �-1,6 branches and is the ma-

jor storage carbohydrate in animals equivalent to starch in plants(1, 2). Glycogen constitutes more than half of the Escherichia colicell mass when the bacteria are grown under conditions of nitro-gen shortage but with an excess carbon source (3–6). Glycogenaccumulation commences during the lag phase, is maximal dur-ing the stationary phase (�15 h), and decreases over the nextseveral hours in many bacteria, such as Bacillus subtilis (7, 8).Glycogen biosynthesis in bacteria and archaea is accomplished byproduction of ADP-glucose and formation of �-1,4 glucosidicchains and �-1,6-linked glucan branches (9), whereas UDP-glu-cose initiates glycogen biosynthesis in mammalian muscle andliver. Thus, the enzyme is involved in glycogen metabolism inbacteria, and archaea share significant homology and enzymaticmechanisms (9). In contrast, breakdown of glycogen is initiatedby debranching enzymes catalyzing hydrolysis of the �-1,6-gluco-sidic linkages of glycogen, amylopectin, and pullulan. Such en-zymes are widely distributed in nature, being found in animals,bacteria (10–14), archaea (15–17), and plants. The glycogen deg-radation process in animals and yeasts is well known, in which thedebranching enzymes exhibit two distinct activities—a glucano-transferase action and amylo-1,6-glucosidase activity (2, 18, 19).However, the glycogen breakdown process is unknown or themode of action of debranching enzymes is not well understood inbacteria and archaea.

One of the main reasons that debranching enzymes of micro-

organisms have been poorly studied is the difficulty in identifyingintermediate reaction products because the substrate (glycogen)has a complex structure, making it difficult to describe the reac-tion mechanism in detail. Thus, we prepared maltodextrin-branched cyclodextrin as a glycogen mimic to study branch chainspecificity of the debranching enzyme. TreX, a glycogen deb-ranching enzyme cloned from the trehalose biosynthesis genecluster of Sulfolobus solfataricus P2, catalyzes the debranching re-action of the branch chain of glycogen into maltodextrin. It be-longs to the GH13 (glycoside hydrolase 13 in the CAZy classifica-tion) family and shows high sequence similarity to isoamylase(20). However, our previous study showed that TreX exhibits bothhydrolysis and transglycosylation activities by catalyzing thebreakage and transfer of �-1,4-glucan oligosaccharides amongchains (20). We also obtained a three-dimensional structure ofTreX and identified two distinct active site configurations, in linewith the bifunctional nature of the enzyme (21). The bifunctionaldebranching enzymes from mammalian tissues transfer the mal-totetraosyl 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, parkkh@snu.ac.kr.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.01442-13

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the branch chain with an �-1,4-glycosidic linkage that is immedi-ately hydrolyzed by glycogen phosphorylase (GlgP). However, un-like the animal debranching enzyme, TreX transfers the glucanmoiety by forming an �-1,6 glycosidic linkage, resulting in trans-location of the branch point within the molecule (20). The break-down processes including the debranching enzyme are likely tohave a different mechanism. Therefore, this study aimed to eluci-date the mechanism of substrate transglycosylation of the TreXenzyme and its effect on the glycogen breakdown process.

MATERIALS AND METHODSMaterials. Thin-layer chromatography (TLC) plates were purchasedfrom Whatman International, Ltd. (Maidstone, Kent, United Kingdom),Ni-nitrilotriacetic acid (NTA) Superflow matrices were obtained fromQiagen (Hilden, Germany), and Bacto tryptone and yeast extract werefrom Difco (Detroit, MI). Other reagents were purchased from SigmaChemical Co. (St. Louis, MO), Merck (Darmstadt, Germany), JunseiChemical Co. (Tokyo, Japan), Showa Chemicals, Inc. (Tokyo, Japan), andWako 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� araD139recA13 �(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) contain-ing ampicillin (100 �g ml�1) at 37°C. TreX tagged with six His residueswas purified on a 1- by 4-cm Ni-NTA column (Ni-NTA Superflow; Qia-gen) as described previously (22), and enzyme purity was confirmed bysodium 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 acetatebuffer (pH 5.5) using 0.5% (wt/vol) amylopectin as the substrate. Thereducing power of hydrolysis products was determined using the dinitro-salicylic acid method (24). One unit of TreX was defined as the amount ofenzyme releasing 1 �mol equivalent of maltose min�1. Protein concen-trations were determined using bovine serum albumin (BSA; SigmaChemical Co.) as a standard (25).

Synthesis of maltodextrin-branched cyclodextrin and branchedmaltoheptaose. Five milliliters of a solution of concentrated maltosyltransferase from Thermatoga maritima (TmMT) was added to a solutionof 5% (wt/vol) maltosyl-�-cyclodextrin (Glc2-�-CD) and 10% (wt/vol)soluble starch in McIlvaine’s buffer (pH 6.5), and the reaction proceededat 70°C for 3 days. The solution was next heated to 100°C for 20 min toinactivate the enzyme and loaded onto a C18 Sep-Pak column to removesmall oligosaccharides. The substrate was purified by several-stage pre-parative high-performance liquid chromatography (HPLC). Dimaltote-traosyl-�-cyclodextrin [(Glc4)2-�-CD] was prepared by a modification ofthe method of Kang et al. (26). To prepare maltotetraosyl-�-1,6 malto-heptaose, 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 debranch-ing enzymes. To clone the GDE of Saccharomyces cerevisiae, four PCRprimers were used: forward1 (5=-CGG GCC ATG GGA ATG AAT AGATCA TTA CTG CTA CGT TTG TCG-3= [NcoI restriction site under-lined]) and reverse1 (TGT CAT AGA TAA GCT TTG TAC AAA CAATGT AGA [HindIII restriction site underlined]) amplifying bases 1 to3123 (the large fragment) and forward2 (TCT ACA TTG TTT GTA CAAAGC TTA TCT ATG ACA [HindIII restriction site underlined]) and re-verse2 (CGC GAA GCT TTC AGG AAT CAT CTT CGT AGG CAT CCCATA A [HindIII restriction site underlined]) amplifying bases 3124 to4608 (the small fragment). These primers were designed with reference tothe full genome sequence in GenBank. The large fragment was PCR am-plified from genomic DNA of S. cerevisiae S288c. The PCR product wasdigested with NcoI and HindIII and ligated with identically cut pPRoEXHTa (Invitrogen, Carlsbad, CA). The recombinant plasmid was used as avector for subsequent cloning of the small fragment, which was amplified

using the forward2 and reverse2 primers followed by HindIII digestion.Finally, the small and large fragments were ligated, and the recombinantgene was sequenced (in pPRoEX Hta). The final recombinant plasmid wasdesignated pPRoEXGDB1.

To overexpress GDB1, pPRoEXGDB1 was transformed into E. coliBL21(DE3). A transformant was cultured in 1 liter LB medium (10 gtryptone, 5 g yeast extract, 5 g sodium chloride) at 37°C with shaking (250rpm), and isopropyl-�-D-thiogalactopyranoside (IPTG) was added to0.01 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 har-vested 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 manufactur-er’s method.

Overexpression and purification of GlgP. The glycogen phosphory-lase gene (glgP) was amplified by PCR from genomic DNA of E. coli K-12,with the addition of restriction sites (underlined below) required for clon-ing. The primers were GlgP/XbaI (5=-CGG AAA AAT CTA GAA CCCTTT GGC CCC GTT C-3=) and GlgP/NdeI (5=-GAA ACG CCC ATA TGAATG CTC CGT TTA CAT ATT C-3=). The PCR product was digested withNdeI and XbaI and ligated into identically cut pTKNd6�H. The resultingconstruct (pTKNd6�H_GlgP) was transformed into E. coli MC1061, andtransformants were cultured on LB medium with kanamycin (50 �gml�1). GlgP was purified by Ni-NTA chromatography, and enzyme ac-tivity on glycogen as the substrate was measured using high-performanceanion-exchange chromatography (HPAEC).

Isolation of glycogen from E. coli. E. coli MC4100 [araD139 deoC1flbB5301 ptsF25 rbsR relA1 rpsL150 �(argF-lac)U169] was cultured in M63minimal salts (1 liter) supplemented with 0.5% (wt/vol) maltose as acarbon source at 37°C for 20 h with shaking (250 rpm). Cells were har-vested by centrifugation (6,000 � g) at 4°C for 20 min, washed withice-cold water, and resuspended in 5 ml 50 mM sodium acetate (pH 4.5).Glycogen was isolated from this suspension and quantified as described byPark 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 (pH6.0). Reactions proceeded for 0.5 and 1 h at 37°C, and products wereanalyzed using TLC and HPAEC.

Preparation of phosphorylase-limited dextrin from glycogen. Phos-phorylase-limited dextrin was prepared as described by Hers et al. (29).Glycogen from E. coli (500 mg) was incubated with 37.5 �g phosphorylasein 20 ml 0.1 M sodium phosphate buffer (pH 6.5) in dialysis tubing anddialyzed against the same buffer. After 24 h, 20 �g phosphorylase a wasadded to the contents of the bag, and the incubation was continued for anadditional 24 h. The reaction was terminated by heating at 100°C for 5min, and insoluble material was removed by centrifugation. The resultingsolution was dialyzed against water and freeze-dried.

Reaction of TreX with branched Glc4-�-CD. TreX (1.5 mU) was in-cubated with Glc4-�-CD (0.5 mM) in 50 mM sodium acetate buffer (pH5.5) 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 for10 min. The samples were centrifuged and analyzed by TLC and HPAEC.

Reaction of glycogen phosphorylase with glycogen and phosphory-lase-limited dextrin from glycogen, in the presence of TreX. Glycogenphosphorylase was incubated with 1% (wt/vol) glycogen or phosphory-lase-limited dextrin in the presence (0.2 to 1 mU) or absence of TreX in 50mM sodium phosphate buffer (pH 6.5) at 37°C. Reactions proceeded for15, 30, and 60 min, and samples were heated at 100°C for 5 min. Insolublematerial was removed by centrifugation, and the resulting solutions wereanalyzed by HPAEC. Glc-1P production was quantified using a standardcurve constructed with the aid of authentic material.

Analysis of reaction products. (i) TLC analysis. Silica gel K5F TLCplates (Whatman) were activated for 1 h at 110°C. Samples were spotted

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onto plates using a pipette, and the plates were placed in a TLC chambercontaining n-butanol– ethanol–water at 5:5:3 (vol/vol/vol). Chromatog-raphy was conducted at room temperature. Reducing sugars were de-tected using the naphthol-sulfuric acid (H2SO4) method (30). Each platewas thoroughly dried and developed by rapid dipping into a methanolsolution containing 3 g N-(1-naphthyl)-ethylene-diamine and 50 ml con-centrated H2SO4 per liter. Each plate was dried and placed in an oven at110°C for 10 min; purple-black spots appeared on a white background.

(ii) HPAEC. The reaction mixtures incubated with TreX were boiledfor 10 min, centrifuged at 12,000 � g for 10 min, and filtered using amembrane filter kit (pore diameter, 0.45 �m) (Millipore, Billerica, MA).Products were applied to a 0.4- by 25-cm CarboPac PA-1 column (Di-onex, Sunnyvale, CA) and analyzed on an HPAEC platform fitted with anED40 pulsed amperometric detector (Dionex). Sixty-microliter aliquotsof sample were injected. Elution was achieved using a NaOAc gradient in150 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 27min, from 300 to 360 mM over 27 to 44 min, and from 360 to 372 mMover 44 to 55 min). The flow rate was 1 ml min�1.

(iii) Isolation and identification of transfer products. TreX (3 mUmg�1 of substrate) was reacted with 0.5% (wt/vol) Glc4-�-CD in 50 mMsodium acetate buffer (pH 5.5) at 70°C for 5 min. The reaction mixturewas next heated at 100°C for 10 min and passed through a 0.45-�m-poreMillex-HV filter (Millipore) prior to two-step HPLC (Jaigel W-251 col-

umn, 2 � 50 cm; W-252 column, 2 � 50 cm) (Japan Analytical Industry,Tokyo, Japan) using distilled water to elute bound materials (flow rate, 3.5ml min�1). Fractions were analyzed by HPAEC. To identify positionalisomers of transfer products, (Glc4)2-�-CD was converted into dimalto-syl-�-cyclodextrin [(Glc2)2-�-CD] by reaction with �-amylase (30 U) at37°C for 12 h, followed by heating at 100°C for 5 min and filtrationthrough a 0.45-�m-pore diameter membrane to improve isomer separa-tion by HPLC. An 1100 HPLC system (Agilent Technologies, Inc., Wald-bronn, Germany) fitted with a Hypercarb column (150- by 4.6-mm inter-nal diameter) (Thermo-Scientific, Loughborough, United Kingdom) andan evaporative light scattering detector (ELSD 2000; Alltech Associates,Inc., Deerfield, IL) was used in the analysis. Elution was performed at 60°Cusing 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 werecalculated using the minimal kinetic scheme for substrate transglycosyla-tion proposed previously by Kawai et al. (31) and Gusakov et al. (32).

Substrate transglycosylation by TreX was considered to proceed asshown in equations 1 and 2 below.

(Glc4)2-�-CD →k6

Glc4-�-CD � Glc4 (2)

Equation 1 yields the rate equations

FIG 1 Thin-layer chromatography analysis of products from reaction mix-tures 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, maltode-caosyl-�-1,6-�-CD; 8, maltotridecaosyl-�-1,6-�-CD; 9, maltotetradecao-syl-�-1,6-�-CD.

FIG 2 Thin-layer chromatography analysis of the reaction products yieldedby TreX acting on 0.5 mM (A) and 0.05 mM (B) Glc4-�-CD. Glc1 to Glc7,standard maltooligosaccharides from glucose to maltoheptaose; �-CD, �-cy-clodextrin; Glc4-�-CD, 6-O-�-maltotetraosyl-�-cyclodextrin; (Glc4)2-�-CD,dimaltotetraosyl-�-cyclodextrin.

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v0

[E]t�

kcat[S] � kcat2[S]2

Km � [S] �[S]2

Km2

(3)

V�-CD �2kcatKm2�S� � kcat2�S�2

KmKm2 � Km2�S� � �S�2 (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 repre-sent the enzyme and the substrate, respectively.

To determine the transglycosylation kinetic parameters, the hydrolyticparameters Km and kcat were measured in reactions with Glc4-�-CD as thesubstrate at 0.05 to 0.3 mM. The transglycosylation kinetic parameterswere determined at higher substrate concentrations (0.5 to 2 mM). Addi-tionally, the first-order reaction rate for hydrolysis of (Glc4)2-�-CD wasseparately determined after isolation of (Glc4)2-�-CD.

RESULTSThe glycogen mimic substrate (Glc4-�-CD) is suitable for char-acterization of the glycogen breakdown process. To validate thesuitability of the glycogen mimic substrate for a glycogen study,Glc4-�-CD was tested with a mouse liver extract and yeast deb-ranching enzymes whose reaction modes are well known. TLC(Fig. 1) and HPAEC (data not shown) yielded information on thereaction products of mouse liver and yeast debranching enzymesacting on Glc4-�-CD (Fig. 1). When the glycogen debranchingenzyme of yeast (GDE) was incubated with Glc4-�-CD, Glc1-�-CD (product no. 3 in Fig. 1A) and a series of transfer prod-ucts—Glc7-�-CD to Glc2-�-CD (no. 6, 7, 8, and 9 in Fig. 1A)—

were obtained. The maltotriosyl moiety of Glc4-�-CD wastransferred to another molecule of Glc4-�-CD and also to othermaterials (Fig. 1). Thus, the GDE of yeast elongated branch chainlength via the formation of �-1,4-glucosidic linkages in the ab-sence 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) wasproduced by GDE action; glucose was released from Glc4-�-CD.However, no (Glc4)2-�-CD was produced when yeast GDE re-acted with Glc4-�-CD. HPAEC and analysis of Rf values by TLCalso identified Glc7-�-CD and Glc1-�-CD. Formation of Glc7-�-CD was confirmed by incubating the putative product withisoamylase; Glc7 and �-CD were produced. These results showthat glycogen degradation by the yeast debranching enzyme can befollowed with Glc4-�-CD as a substrate analog.

TreX has both hydrolytic and transglycosylation activity athigh substrate concentrations (>0.5 mM). The enzyme was in-cubated with Glc4-�-CD (0.5 mM) in 50 mM sodium acetate buf-fer (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°Cfor 10 min prior to analysis by TLC and HPAEC. Unlike what wasnoted when the debranching enzymes of yeast and mouse liverextract were studied, TreX did not generate �-1,4 hydrolysis prod-ucts 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 reactiontime was extended (Fig. 2, 3, and 4). When the Glc4-�-CD con-

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;3, mixture of isomers of dimaltotetraosyl-1,6-�-CD; 4, maltotetraose.

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centration was 0.05 mM, the Glc4-�-CD level decreased gradually,whereas the �-CD and maltotetraose concentrations increasedwith the reaction time (Fig. 2B to 4B). As the positional isomers ofthe (Glc4)2-�-CD transfer products were not separated on theHypercarb column, they were first converted to (Glc2)2-�-CD by�-amylase (Fig. 5A and B) and analyzed by HPLC using the Hy-percarb column. As shown in Fig. 5C, comparison of the chro-matographic profile with that of (Glc2)2-�-CD reference materials(21) indicated that the transfer product was a mixture of 61,63-and 61,64-(Glc4)2-�-CD at a molar ratio close to unity. Addition-ally, the formation of 61,64-(Glc4)2-�-CD observed in the presentstudy and the synthesis of 61,64-(Glc2)2-�-CD reported by Kang etal. (26) support the idea that the transfer products produced frombranch chains varying widely in size are similar.

Kinetics of hydrolysis and transglycosylation of Glc4-�-CDby TreX. The enzyme was added to Glc4-�-CD solutions of vari-ous concentrations (0.05 to 2 mM). As transglycosylation was ob-served at substrate concentrations of 0.5 mM (Fig. 2 and 3), akinetic analysis was conducted using two ranges of substrate con-

centration. The Km and kcat of hydrolysis were assessed at substrateconcentrations of 0.05 to 0.3 mM, and the Km2 and kcat2 of trans-glycosylation were determined at 0.5 to 2 mM. Data plottingyielded the initial velocities of Glc4 production at various substrateconcentrations. A Lineweaver-Burk plot (data not shown) showedthat 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 pa-rameters of transglycosylation, TreX was reacted with Glc4-�-CDat 0.5 to 2 mM. The formation of �-CD was quantified by HPAECto obtain initial reaction velocities. Equation 4 was used to esti-mate 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 occurredprincipally at high substrate concentrations. The branched glyco-syl chain of the Glc4 substrate was transferred to a hydroxyl groupof an acceptor molecule other than water, and the extent of hydrolysiswas reduced. Thus, transglycosylation may influence the overall re-action rate. As the reaction proceeds, hydrolysis began to dominateover transglycosylation, leading to the production of Glc4 and �-CDfrom the TreX transglycosylation products (Fig. 4).

TreX has high specific activity for a wide range of branchchain lengths. Glycogen contains branch chains of various

FIG 4 Time course analysis of TreX products produced when the enzymeacted on 0.5 mM (A) and 0.05 mM (B) Glc4-�-CD.�, maltotetraose; ●, �-CD;�, maltotetraosyl-1,6-�-CD; �, dimaltotetraosyl-�-CD.

FIG 5 HPAEC analysis (A and B) of transfer products prior to �-amylasetreatment (A) and after �-amylase treatment (B). The positional isomers of(B) were analyzed by HPLC using a Hypercarb column (C). Products: 1, amixture of 61,63- and 61,64-dimaltotetraosyl-�-CD; 2, a mixture of 61,63-and 61,64-dimaltosyl-�-CD; 2A, 61,63-dimaltosyl-�-CD; 2B, 61,64-di-maltosyl-�-CD.

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lengths. To explore TreX activities on various branch chains, Glc2-to Glc10-�-CDs were incubated with TreX, and the products wereanalyzed by HPAEC and TLC. Unexpectedly, TreX was active onall branch chains studied and the kcat/Km ratio increased as thebranch chain degree of polymerization (DP) rose (Fig. 6).

Action of TreX on branched maltotetraosyl-�-1,6-malto-heptaose. Unlike cyclodextrin, glycogen is a linear glucan. To con-firm that TreX was active on linear glucans, the enzyme was incu-bated with maltotetraosyl-�1,6-maltoheptaose, yielding threemajor products after a 10-min reaction. The substrate was hydro-lyzed to maltoheptaose and maltotetraose. In parallel, the malto-tetraose moiety was transferred to another substrate molecule,producing dimaltotetraosyl maltoheptaose, which was in turn

further hydrolyzed to maltotetraose and maltoheptaose (data notshown). This supports the suggestion that TreX exerts �-1,6-transfer activity on branched glucans. The reaction did not yieldeither maltooctaose (Glc8) or maltoundecaose (Glc11), indicatingthat the maltotetraosyl moiety was not transferred via formationof an �-1,4 linkage but rather an �-1,6 linkage.

Transglycosylation enhances the rate of glycogen dephos-phorylation. To explore the effect of transglycosylation on glyco-gen degradation in a model system, glycogen (E. coli MC4100) wasincubated with glycogen phosphorylase in the presence or absenceof TreX. The latter enzyme significantly increased the rate ofGlc-1P formation (5.48 � 10�3 mmol·liter�1·min�1 compared tothe control value of 3.91 � 10�3 mmol·liter�1·min�1). Glc-1P wasformed more rapidly when glycogen was present at 1.0 mM thanthat at 0.5 mM (Table 1), which was in line with the observationthat transglycosylation occurred preferentially at higher concen-trations of Glc4-�-CD (Fig. 2 and 3). In contrast, TreX activity onlimited dextrin did not vary with substrate concentration (Table1). A slight increase in the rate in the absence of TreX was likelydue to the incomplete limited dextrin preparation. These resultsindicate that shifting branch chains to the outer layers of glycogenwas achieved via transglycosylation, thus, increasing the numberof nonreducing glucose moieties available to phosphorylase(Table 1).

DISCUSSION

Branched cyclodextrins as substrate mimics were used previouslyto characterize yeast debranching enzymes (33). However, trans-fer products were only indirectly identified by further hydrolysiswith pullulanase, in which dibranched products cannot be de-tected 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. Thisfinding suggested that TreX transfers the maltosyl residue of Glc2-�-1,6-�-CD with formation of an �-1,6-glycosidic linkage. TreXalso yielded (Glc4)2-�-CD containing an �-1,6-glucosidic linkage(Fig. 7). Similarly, 63-maltotetraosyl maltoheptaose, similar to aglycogen branch, produced the transfer product of (Glc4)2-malto-heptaose (data not shown). Thus, transglycosylation occurs viaformation of an �-1,6-glycosidic linkage when either branchedlinear maltodextrin or Glc4-�-CD serves as the substrate (data not

FIG 6 TreX activities toward various branched cyclodextrins: Glc2-�-CD (6-O-�-maltosyl-�-cyclodextrin), Glc4-�-CD (6-O-�-maltotetraosyl-�-cyclo-dextrin), Glc6-�-CD (6-O-�-maltohexaosyl-�-cyclodextrin), and Glc8-�-CD(6-O-�-maltooctaosyl-�-cyclodextrin). (A) TLC analysis of the substrate con-centration at various reaction times; (B) values of the kcat/Km ratio.

TABLE 1 Effect of TreX on formation of glucose-1-phosphatea

SubstrateTreXconcn (mU)

Glucose-1-phosphate concn (mM) at:Rate constant (mmolliter�1·min�1) � 10�315 min 30 min 60 min

Limited dextrin, 10 mg/ml 0.0 0.030 � 0.002 0.052 � 0.004 0.071 � 0.058 1.15 � 0.090.2 0.030 � 0.002 0.049 � 0.003 0.067 � 0.042 1.08 � 0.070.5 0.028 � 0.002 0.048 � 0.003 0.064 � 0.040 1.04 � 0.06

Glycogen1 mM 0.0 0.086 � 0.006 0.137 � 0.011 0.242 � 0.019 3.91 � 0.31

0.2 0.092 � 0.003 0.180 � 0.012 0.331 � 0.022 5.48 � 0.360.5 0.092 � 0.001 0.171 � 0.002 0.323 � 0.005 5.71 � 0.39

0.5 mM 0.0 0.045 � 0.004 0.062 � 0.05 0.132 � 0.012 2.13 � 0.190.2 0.045 � 0.004 0.078 � 0.08 0.148 � 0.016 2.44 � 0.130.5 0.045 � 0.012 0.075 � 0.20 0.145 � 0.039 2.38 � 0.16

a 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.

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shown). Additionally, transglycosylation of branch chains can oc-cur between two molecules or within a molecule. Therefore, gly-cogen consisting of a large number of branch chains in a moleculepossibly includes a transglycosylation step in which the same gly-cogen molecule has both acceptor and donor sites. The branchchains occupy �62% of the space within a glycogen molecule,indicating an extremely high density between branch chains inglycogen (34). Therefore, we assume that the local concentrationsof donors and acceptors within an individual glycogen moleculemay be sufficiently high to trigger transglycosylation. This obser-vation indicates that TreX exerts transfer activity, forming �-1,6-glycosidic linkages in the outer layers of glycogen. In addition, theextent of transglycosylation is probably much higher when au-thentic glycogen is the substrate, rather than artificial branched�-CDs. Tabata et al. observed that the transferase activity of yeastdebranching enzyme acting on limited dextrin was higher thanthat when a substrate mimic, Glc4-�-CD, was employed (33).

The substrate specificity to various branch chain lengths ofglycogen was determined in detail to determine which branchchain could be efficiently employed during glycogen breakdown.Possible transglycosylation reactions of microbial debranchingenzymes require more intensive study, as the enzymes of archaeaand bacteria have received little attention compared to those ofanimals and yeasts. A previous study indicated that TreX accepts awide spectrum of substrates, the chains of which have DP values of3 to 6 (23). In the present study, we further analyzed the effects ofbranch chain length using a wide range of substrates. Unexpect-edly, TreX was active on branched chains of DP2 to DP10; transferproducts were evident with all tested substrates. This result agreeswith the suggestion of Woo et al. (21) that subunit tetramerizationgenerates a channel-like cavity that allows long branch chains tobe accommodated. An isoamylase from Pseudomonas spp. had thehighest specific activity toward a branch chain of DP7, but deb-ranching of the DP2 chain was minimal (data not shown). Thedebranching enzyme from Nostoc punctiforme exhibits a catalytic

preference for longer maltooligosaccharides (DP, 8) (11). Incontrast, GlgX, a debranching enzyme from E. coli, is active mainlyagainst DP4 chains (5, 10), and AmyX from B. subtilis is specificfor DP3 to -6 chains (7). A substrate with a branch chain length ofDP4 can be considered a suitable substrate for kinetic analysisbecause such a substrate is within the range of preferable chainlength for TreX. Moreover, a limited dextrin containing a branchchain of DP4 is produced via the action of glycogen phosphorylaseand is also conveniently studied in relation to mammalian deb-ranching enzyme. The average branch chain lengths of glycogen inE. coli (28), S. solfataricus (35), and B. subtilis (36) are DP7 to -11,and the structures are expected to be similar to that of mammalianglycogen (DP13). Meléndez-Hevia et al. (34) and Meléndez et al.(37) developed a mathematical model of the mammalian glycogenmolecule. An optimized glycogen structure features chains thatare an average of 13 glucose residues in length. The chains areexposed to the action of phosphorylase because they are suffi-ciently flexible to allow binding to the enzyme active site. More-over, the presence of a large number of such chains ensures that aphosphorylase active site rendered vacant by loss of a chain can beimmediately filled by another adjacent chain (34).

Branch chains on the glycogen surface are accessible by phos-phorylase. The three-dimensional structure of mammalian glyco-gen phosphorylase features a narrow 30-Å crevice that binds gly-cogen, 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 samesurface. A consequence of transglycosylation of branch chains isan increase in chain density on the outer glycogen layer. Internalchains are shifted outward, increasing the number of nonreducingends available to phosphorylase (Fig. 8). The kinetic data (Table 1)revealed that such outward transfer provides many points of at-tack for phosphorylase, increasing glucose release. As shown inFig. 2A, transglycosylation occurs preferentially at high substrateconcentrations (�0.5 mM Glc4-�-CD). Branch chains occupy

FIG 7 Schematic diagram of hydrolysis and transglycosylation of maltotetraosyl �-cyclodextrin (Glc4-�-CD) catalyzed by TreX.

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�62% of the space within a glycogen molecule, indicating an ex-tremely high density between branch chains in glycogen (34).Therefore, we can assume that the local concentrations of branchchains in the outer tier of the glycogen molecule may be suffi-ciently high to enable transglycosylation.

Glycogen metabolism is highly interconnected with a wide va-riety of cellular processes, including stringent response, Mg2

availability, and osmotic pressure (40). Therefore, glycogen as areserve macromolecule has little effect on the internal osmoticpressure of the cell (2). Maintaining osmotic pressure duringthe glycogen degradation is important. Instead of hydrolysis of thebranch chain, in this model, transglycosylation may maintain thelevel of osmotic pressure and fluid viscosity, as the release of linearbranch chains (DPs of 5 to 12) by hydrolysis may cause a drasticincrease in osmotic pressure and viscosity of cellular fluid,whereas Glc-1P from the transferred branch chains can be imme-diately metabolized into the glycolytic pathway without a signifi-cant change in osmotic pressure. A mutant enzyme that has highhydrolysis activity with low transglycosylation activity or cloningof a novel debranching enzyme from another microbial sourcehaving low transglycosylation selectivity is required for furtherstudy. Moreover, transglycosylation inhibits hydrolysis (31). De-pending on the environment of a given microorganism, deb-ranching (hydrolysis) of glycogen may be controlled by productinhibition and may be further hindered by transglycosylation sidereactions. Until recently, the physiological function of the micro-bial debranching enzyme has been considered to be principallyhydrolysis. However, we suggest that transglycosylation may alsobe involved in glycogen degradation. Similarly, trehalose biosyn-thesis in Sulfolobus may proceed via transglycosylation catalyzedby TreX in association with the actions of enzymes encoded bytreZY (15, 16), and glucan chains shifted to the outer layer by TreXmay facilitate the actions of maltooligosyl trehalose synthase(TreY) and maltooligosyl trehalose hydrolase (TreZ). We haveshown that just as mammalian debranching enzymes exert dualfunctions, transglycosylation in archaea may assist in glycogenbreakdown by forming and shifting �-1,6-glucosidic linkages bychain transglycosylation to the outer layers of glycogen. However,in the present study, we used kinetic data to explore the possiblecontribution of transglycosylation to glycogen breakdown in amodel system in which TreX from S. solfataricus was introduced toglycogen and glycogen phosphorylase from E. coli. Therefore, a

full set of enzymes involved in the glycogen metabolism of a strainof bacteria or archaea should be introduced, and their actionsshould be analyzed in concert.

ACKNOWLEDGMENTS

This study was supported by the Basic Research Program of the NationalResearch Foundation (grant no. 2012R1A1A2005012) and the Next Gen-eration BioGreen21 Program (SSAC, grant no. PJ009086), Rural Devel-opment Administration, Republic of Korea.

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