FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091551142> ?p ?o ?g. }

• W2091551142 endingPage "15733" @default.
• W2091551142 startingPage "15729" @default.
• W2091551142 abstract "The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilicBacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic α-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known α-, β-, and γ-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic α-1,4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic α-1,4-glucans and into the final major product, β-CD. CGTase from Bacillus maceransalso produced large cyclic α-1,4-glucans except that the final major product was α-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase. The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilicBacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic α-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known α-, β-, and γ-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic α-1,4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic α-1,4-glucans and into the final major product, β-CD. CGTase from Bacillus maceransalso produced large cyclic α-1,4-glucans except that the final major product was α-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase. Cyclodextrin glucanotransferase (CGTase, 1The abbreviations used are: CGTase, cyclodextrin glucanotransferase; CD, cyclodextrin; DP, degree of polymerization; D-enzyme; disproportionating enzyme; HPAEC, high performance anion exchange chromatography; HPLC, high performance liquid chromatography; TOF-MS, time of flight mass spectrometry. 1The abbreviations used are: CGTase, cyclodextrin glucanotransferase; CD, cyclodextrin; DP, degree of polymerization; D-enzyme; disproportionating enzyme; HPAEC, high performance anion exchange chromatography; HPLC, high performance liquid chromatography; TOF-MS, time of flight mass spectrometry. EC 2.4.1.19), found in several bacterial species, catalyzes the inter- and intramolecular transglycosylation of α-1,4-glucan. Such activity of CGTase on inter- and intramolecular transglycosylation of α-1,4-glucan is called the disproportionation reaction and the cyclization reaction, respectively. It is also known that CGTase catalyzes the transglycosidic linearization (coupling reaction) of cyclic α-1,4-glucan in the presence of a suitable acceptor molecule to produce linear α-1,4-glucan. The cyclization reaction of CGTase has been of great interest since this is the only enzyme that can produce α-, β- and γ-cyclodextrin (CD), which are generally known as the cyclic α-1,4-glucan with DP of 6, 7, or 8. These CDs all have a hydrophobic central cavity, incorporate various inorganic or organic compounds, and form inclusion complexes (1Saenger W. Angew. Chem. Int. Ed. 1980; 19: 344-362Crossref Scopus (2034) Google Scholar). Therefore, these CDs are widely used in the pharmaceutical, food, agricultural, and cosmetic industries (2Schmid G. Trends Biotechnol. 1989; 7: 244-248Abstract Full Text PDF Scopus (115) Google Scholar). Extensive analyses on various CGTases indicated that all CGTases convert amylose or amylopectin into a mixture of α-, β-, and γ-CD and remaining dextrins; differences, however, were found in their product specificities (α-, β-, and γ-CD ratios). Thus, CGTase is sometimes classified into three types (α-, β-, and γ-CGTase), depending on the major CD produced. Since α-, β-, and γ-CD all have a dimensionally distinct central cavity and different specificity for guest molecules, recent studies on CGTase have focused on trying to understand the mechanism of the cyclization reaction and to find or engineer a CGTase that produces a specific type of CD. Several approaches have been carried out to obtain the structural explanation of the cyclization reaction of CGTase. Analyses of the three-dimensional structure of CGTase have been carried out using several types of CGTases (3Klein C. Schulz G.E. J. Mol. Biol. 1991; 217: 737-750Crossref PubMed Scopus (214) Google Scholar, 4Knegtel R.M.A. Wind R.D. Rozeboom H.J. Kalk K.H. Buitelaar R.M. Dijkhuizen L. Dijkstra B.W. J. Mol. Biol. 1996; 256: 611-622Crossref PubMed Scopus (85) Google Scholar, 5Kubota M. Matsuura Y. Sakai S. Katsube Y. Oyo Toshitsu Kagaku. 1994; 41: 245-253Google Scholar, 6Lawson C.L. van Montfort R. Strokopytov B. Rozeboom H.J. Kalk K.H. de Vries G.E. Penninga D. Dijkhuizen L. Dijkstra B.W. J. Mol. Biol. 1994; 236: 590-600Crossref PubMed Scopus (221) Google Scholar). Additionally, the structures of CGTases with substrates (5Kubota M. Matsuura Y. Sakai S. Katsube Y. Oyo Toshitsu Kagaku. 1994; 41: 245-253Google Scholar, 7Klein C. Hollender J. Bender H. Schulz G.E. Biochemistry. 1992; 31: 8740-8746Crossref PubMed Scopus (105) Google Scholar, 8Knegtel R.M.A. Strokopytov B. Penninga D. Faber O.G. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. J. Biol. Chem. 1995; 270: 29256-29264Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 9Strokopytov B. Knegtel R.M.A. Penninga D. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. Biochemistry. 1996; 35: 4241-4249Crossref PubMed Scopus (136) Google Scholar) and with inhibitor molecules (9Strokopytov B. Knegtel R.M.A. Penninga D. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. Biochemistry. 1996; 35: 4241-4249Crossref PubMed Scopus (136) Google Scholar, 10Strokopytov B. Penninga D. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. Biochemistry. 1995; 34: 2234-2240Crossref PubMed Scopus (136) Google Scholar) were also analyzed. From these studies, models of CGTase activity cleaving the target α-1,4-linkage were proposed. In the case of CGTase from Bacillus circulans strain 251, three active site residues, Asp-229, Glu-257, and Asp-328, which are conserved in all CGTase primary sequences, play important roles for this step of reaction (8Knegtel R.M.A. Strokopytov B. Penninga D. Faber O.G. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. J. Biol. Chem. 1995; 270: 29256-29264Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 10Strokopytov B. Penninga D. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. Biochemistry. 1995; 34: 2234-2240Crossref PubMed Scopus (136) Google Scholar). A similar catalytic mechanism has been reported from structural studies on amylases (11Buisson G. Duée E. Haser R. Payan F. EMBO J. 1987; 6: 3909-3916Crossref PubMed Scopus (352) Google Scholar, 12Larson S.B. Greenwood A. Cascio D. Day J. McPherson A. J. Mol. Biol. 1994; 235: 1560-1584Crossref PubMed Scopus (156) Google Scholar, 13Matsuura Y. Kusunoki M. Harada W. Kakudo M. J. Biochem. ( Tokyo ). 1984; 95: 697-702Crossref PubMed Scopus (601) Google Scholar, 14Qian M. Haser R. Buisson G. Duée E. Payan F. Biochemistry. 1994; 33: 6284-6294Crossref PubMed Scopus (279) Google Scholar) that hydrolyze α-1,4-glucan. Several amino acid residues involved in substrate binding or in the determination of product specificity have also been proposed by three-dimensional structure analysis (5Kubota M. Matsuura Y. Sakai S. Katsube Y. Oyo Toshitsu Kagaku. 1994; 41: 245-253Google Scholar, 6Lawson C.L. van Montfort R. Strokopytov B. Rozeboom H.J. Kalk K.H. de Vries G.E. Penninga D. Dijkhuizen L. Dijkstra B.W. J. Mol. Biol. 1994; 236: 590-600Crossref PubMed Scopus (221) Google Scholar, 8Knegtel R.M.A. Strokopytov B. Penninga D. Faber O.G. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. J. Biol. Chem. 1995; 270: 29256-29264Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 9Strokopytov B. Knegtel R.M.A. Penninga D. Rozeboom H.J. Kalk K.H. Dijkhuizen L. Dijkstra B.W. Biochemistry. 1996; 35: 4241-4249Crossref PubMed Scopus (136) Google Scholar) or by protein engineering approaches (15Nakamura A. Haga K. Yamane K. Biochemistry. 1993; 32: 6624-6631Crossref PubMed Scopus (97) Google Scholar, 16Nakamura A. Haga K. Yamane K. Biochemistry. 1994; 33: 9929-9936Crossref PubMed Scopus (77) Google Scholar, 17Penninga D. Strokopytov B. Rozeboom H.J. Lawson C.L. Dijkstra B.W. Bergsma J. Dijkhuizen L. Biochemistry. 1995; 34: 3368-3376Crossref PubMed Scopus (147) Google Scholar). However, it is less clearly understood how CGTase catalyzes the following intramolecular transfer reaction to produce cyclodextrins. Although α-, β-, and γ-CD are the major products of CGTase, it has been known that trace amounts of larger cyclic glucans (δ-, ε-, ζ-, η-, and θ-CD) were also present in the reaction mixture of CGTase on starch (18French D. Pulley A.O. Effenberger J.A. Rougvie M.A. Abdullah M. Arch. Biochem. Biophys. 1965; 111: 153-160Crossref PubMed Scopus (117) Google Scholar, 19Pulley A.O. French D. Biochem. Biophys. Res. Commun. 1961; 5: 11-15Crossref PubMed Scopus (57) Google Scholar). The structures of these larger cyclic glucans are still not well understood, because they seem to be a mixture of cyclic α-1,4-glucans, outer-branched cyclic α-1,4-glucans, and inner-branched cyclic glucans (18French D. Pulley A.O. Effenberger J.A. Rougvie M.A. Abdullah M. Arch. Biochem. Biophys. 1965; 111: 153-160Crossref PubMed Scopus (117) Google Scholar). Kobayashi and Yamasaki (20Kobayashi S. Yamazaki M. Denpun Kagaku. 1991; 38: 314Google Scholar) carried out further structural analyses on putative δ-, ε-, ζ-, η-, and θ-CD fractions and reported that the δ-CD fraction contained a large amount of unbranched cyclic α-1,4-glucan with DP 9. However, the proportion of unbranched cyclic α-1,4-glucan in the following fractions decreased dramatically (50% in ε-CD, 25% in ζ-CD, and almost 0% in η- and θ-CD fractions). From this study, it is thought that the presence of cyclic α-1,4-glucan with DP larger than 12 in the CGTase reaction products is unlikely. Recently, however, we found that potato D-enzyme (disproportionating enzyme or 4-α-glucanotransferase, EC 2.4.1.25) catalyzes an intramolecular transglycosylation reaction on amylose to produce cyclic α-1,4 glucans with DP range from 17 to several hundred (21Takaha T. Yanase M. Takata H. Okada S. Smith S.M. J. Biol. Chem. 1996; 271: 2902-2908Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The time-course analysis of D-enzyme action on amylose revealed that large cyclic α-1,4-glucans were preferentially produced in the initial stage of the cyclization reaction, and subsequently converted into small cyclic α-1,4-glucans, although α-, β-, and γ-CD were never produced (21Takaha T. Yanase M. Takata H. Okada S. Smith S.M. J. Biol. Chem. 1996; 271: 2902-2908Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). D-enzyme also catalyzes disproportionating reactions on malto-oligosaccharides (22Takaha T. Yanase M. Okada S. Smith S.M. J. Biol. Chem. 1993; 268: 1391-1396Abstract Full Text PDF PubMed Google Scholar) and transglycosidic linearization of cyclic α-1,4-glucans in the presence of a suitable acceptor (21Takaha T. Yanase M. Takata H. Okada S. Smith S.M. J. Biol. Chem. 1996; 271: 2902-2908Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In all these respects, D-enzyme and CGTase both seem to catalyze the same reaction, with the major difference being the DP of the cyclic α-1,4-glucan produced. Furthermore, we also reported that the glycogen-branching enzyme (EC 2.4.1.18) from Bacillus stearothermophilus also catalyzes the intramolecular transglycosylation of amylose and amylopectin to produce branched cyclic glucans with DP larger than 18 (23Takata H. Takaha T. Okada S. Takagi M. Imanaka T. J. Bacteriol. 1996; 178: 1600-1606Crossref PubMed Google Scholar, 24Takata H. Takaha T. Okada S. Hizukuri S. Takagi M. Imanaka T. Carbohydr. Res. 1996; 295: 91-101Crossref PubMed Google Scholar). From these studies on glycosyltransferases other than CGTase, we are interested in the action of CGTase in producing cyclic glucans larger than CDs. In this paper, we investigated the initial action of CGTase from an alkalophilicBacillus sp. A2-5a (25Kometani T. Terada Y. Nishimura T. Takii H. Okada S. Biosci. Biotechnol. Biochem. 1994; 58: 517-520Crossref Scopus (33) Google Scholar) on synthetic amylose and found that the CGTase also produced cyclic α-1,4-glucans with DP ranging from 9 to more than 60, in addition to α-, β-, and γ-CD, from synthetic amylose in the very early stage of the reaction. Synthetic amylose with an average molecular mass of 30 kDa (amylose AS-30) and soluble starch were purchased from Nakano Vinegar Co., Ltd. (Aichi, Japan) and E. Merck AG (Darmstadt, Germany), respectively. Glucoamylase fromRhizopus sp. was purchased from Toyobo Co., Ltd. (Osaka, Japan). CGTase from an alkalophilic Bacillus sp. A2-5a was purified to a homogeneous state (25Kometani T. Terada Y. Nishimura T. Takii H. Okada S. Biosci. Biotechnol. Biochem. 1994; 58: 517-520Crossref Scopus (33) Google Scholar). CGTases from B. macerans was purchased from Amano Pharmaceutical Co., Ltd. (Aichi, Japan) and was used without further purification. The activity of CGTase was assayed using soluble starch as the substrate by measuring the decrease in iodine-staining power as described previously (25Kometani T. Terada Y. Nishimura T. Takii H. Okada S. Biosci. Biotechnol. Biochem. 1994; 58: 517-520Crossref Scopus (33) Google Scholar). Amylose AS-30 (8 mg) was dissolved in 200 μl of 1 n NaOH solution then neutralized by adding 200 μl of 1 m sodium acetate buffer (pH 5.5), 200 μl of 1 n HCl, and 400 μl of distilled water. The solution was used immediately after neutralization. CGTase (0.75 unit/ml) was incubated at 40 °C with amylose AS-30 (0.4% (w/v)) in 0.2 m sodium acetate buffer, pH 5.5, and reactions were terminated by boiling the solutions for 10 min. The reaction mixture containing 20 μg of glucan was incubated with glucoamylase (0.2 units) in 20 mm sodium acetate buffer (pH 5.5) for 16 h at 40 °C and then boiled for 5 min. The products in the reaction mixture were determined with high performance anion exchange chromatography (HPAEC, see below). The amounts of α-, β-, and γ-CD were measured with high performance liquid chromatography (HPLC, see below). The amount of glucoamylase-resistant molecules was calculated by subtracting the amount of glucose released by glucoamylase from that of total glucan in the reaction mixture. The amount of glucose was measured by the glucose oxidase method (26Miwa I. Okuda J. Maeda K. Okuda G. Clin. Chim. Acta. 1972; 37: 538-540Crossref PubMed Scopus (271) Google Scholar). HPAEC was carried out based on the DX-300 system (Dionex) with a pulsed amperometric detector (model PAD-II, Dionex) using a Carbopac PA-100 column (4 mm × 250 mm). A sample (25 μl) containing 40 μg of glucan was injected and eluted with a gradient of sodium acetate (0–2 min, 50 mm; 2–37 min, increasing from 50 mm to 350 mm with the installed gradient program 3; 37–45 min, increasing from 350 mm to 850 mm with the installed gradient program 7; 45–47 min, 850 mm) in 150 mm NaOH with a flow rate of 1 ml min−1. HPLC was carried out based on the DX-300 system (Dionex) using an Aminex HPX-42A column. To remove glucose from the reaction mixtures, a sample (50 μl) containing 80 μg of glucan was charged on a Waters Sep-Pak C18 cartridge (Millipore), washed with 10 ml of H2O and eluted with 1.5 ml of 50% methanol. The eluate was dried up in vacuo, and dissolved in 50 μl of water. The sample was then injected and eluted with water with a flow rate of 0.6 ml min−1 at 80 °C. The eluate from the column was mixed with 0.3 m LiOH using an anion micromembrane suppressor (model AMMS-II, Dionex), after which the carbohydrate in the eluate was detected with a pulsed amperometric detector (mode PAD-II, Dionex). A reaction mixture (5 ml) containing 10 mg of amylose AS-30 and CGTase (0.7 unit) was incubated in 0.2 m sodium acetate buffer, pH 5.5, at 40 °C for 1 h, and then boiled for 10 min. The reaction mixture was incubated with 10 units of glucoamylase at 40 °C for 16 h, and then boiled for 5 min. After removing glucose with the Waters Sep-Pak C18 cartridge, the molecular masses of glucoamylase-resistant glucans were determined with a Kompact Maldi I TOF-MS system (Shimadzu, Kyoto, Japan). Synthetic amylose AS-30 was incubated with CGTase from alkalophilic Bacillus sp. A2-5a. The enzyme reaction was terminated at the early stage of the reaction (10 min), and then the reaction mixture was incubated with glucoamylase to digest the linear amylose into glucose. When the glucoamylase-resistant molecules thus obtained were analyzed with HPAEC, many peaks were detected (Fig.1 A). Note that these peaks were not found in a control experiment with heat-inactivated CGTase (result not shown). Most of these peaks were eluted in the region where cyclic α-1,4-glucans with DPs of over 17, produced by potato D-enzyme on synthetic amylose (Fig. 1 F), were eluted. This result suggests that CGTase produced such cyclic α-1,4-glucans. The large peak around 42 min may indicate the presence of glucoamylase-resistant molecules with DP more than 60, since these molecules were not resolved in this HPAEC condition and eluted together. Then to prove the cyclic structure of glucoamylase-resistant molecules produced by CGTase, their molecular masses were determined with TOF-MS (Fig. 2). A non-cyclic glucan with DP of nhas a molecular mass of 162.1436n + 18.01534 Da, whereas a cyclic glucan should have a molecular mass of 162.1436n Da. The molecular mass of each glucoamylase-resistant molecule was compared with the theoretical value for non-cyclic and cyclic glucans (TableI). As shown in Table I, experimental values of glucoamylase-resistant molecules were consistent with theoretical values for cyclic glucan but not with those for non-cyclic glucan.Table IExperimental and theoretical masses of glucansDPExperimentalTheoreticalCyclic glucanNoncyclic glucan699599610147115711581176813191320133891483148215001016431644166211180718071825121969196919871321312131214914229222932311152456245524731626172617263517277827792797182942294229601931043104312220326732663284Experimental masses of glucans were determined by TOF-MS. Theoretical masses of cyclic and noncyclic glucans were calculated as 162.1436n + 22.9898 and 162.1436n + 22.9898 + 18.01534, respectively. Values are rounded and presented as whole numbers. 162.1436, the mass of glucosyl residue; n, DP; 22.9898, the mass of sodium ion; 18.01534, the mass of H2O. Open table in a new tab Experimental masses of glucans were determined by TOF-MS. Theoretical masses of cyclic and noncyclic glucans were calculated as 162.1436n + 22.9898 and 162.1436n + 22.9898 + 18.01534, respectively. Values are rounded and presented as whole numbers. 162.1436, the mass of glucosyl residue; n, DP; 22.9898, the mass of sodium ion; 18.01534, the mass of H2O. To confirm that the glucoamylase-resistant molecules produced by CGTase are α-1,4-glucans, the structure of these glucans were further examined by treatment with several enzymes. α-Amylase fromBacillus subtilis, an endo-type amylase, completely degraded these molecules to glucose and maltose, and isoamylase and pullulanase, which degrade α-1,6-linkage of glucans, did not (data not shown). These results indicate that the glucoamylase-resistant molecules produced by CGTase were α-1,4-glucans. Based on all the results mentioned above, we concluded that the molecules produced by the CGTase reaction on amylose, as shown in Fig.1 A, were cyclic α-1,4-glucans with DPs ranging from 6 to more than 60. It is known that the glucoamylase-resistant products of the CGTase reaction on amylose, after a prolonged reaction time, are α-, β-, γ-CD and negligible amounts of other glucoamylase-resistant glucans. Our result apparently contradicts this widely held view since the major cyclic α-1,4-glucans produced in the initial stage of the CGTase reaction were not α-, β-, and γ-CD but were those with high DPs. To investigate how large cyclic α-1,4-glucans, which were found in the initial stage of CGTase reaction, were replaced by α-, β-, and γ-CD, the time course of the reaction of CGTase were monitored. Amylose AS-30 was incubated with CGTase from alkalophilicBacillus sp. A2-5a for up to 360 min. Each sample was treated with glucoamylase and was analyzed by HPAEC (Fig. 1). As shown in Fig. 1 (A–E), cyclic α-1,4-glucans with DPs of over 60 were most prevalent after a reaction time of 10 min (Fig.1 A). However, with prolonged reaction, cyclic α-1,4-glucans with high DPs gradually decreased, and those with low DPs increased. At the end of the reaction, the main product of CGTase was β-CD (Fig. 1 E). Fig. 3 shows the time course of the amount of cyclic α-1,4-glucans. The amount of total cyclic α-1,4-glucans increased rapidly from the beginning of the reaction, and their yield reached about 80% at 120 min. However, the amounts of α-, β-, and γ-CD increased at a more slower rate than those of total cyclic α-1,4-glucans. As a result, cyclic α-1,4-glucans, apart from α-, β-, and γ-CD, increased at the early stage of the reaction and reached a yield of about 52% at 50 min, but decreased thereafter. CGTases found in many bacterial species are classified into three types, α-, β-, or γ-CGTase, depending on the major product of the cyclization reaction. The CGTase employed above is β-CGTase because it mainly produces β-CD (25Kometani T. Terada Y. Nishimura T. Takii H. Okada S. Biosci. Biotechnol. Biochem. 1994; 58: 517-520Crossref Scopus (33) Google Scholar). To examine whether the production of a large cyclic α-1,4-glucan is the specific feature only found in this CGTase or is the common feature for others, similar experiments were carried out by using CGTases from B. macerans, which is classified as α-CGTase. This enzyme also produced large cyclic α-1,4-glucans in the initial stage of reaction (Fig. 4, Aand B), which were subsequently converted into small cyclic α-1,4-glucans. However, the final major cyclic product was α-CD (Fig. 4 E). It is generally believed that the cyclization reaction of CGTase on amylose is an exo-type attack (2Schmid G. Trends Biotechnol. 1989; 7: 244-248Abstract Full Text PDF Scopus (115) Google Scholar), where the enzyme recognizes the 6–8 glucose units from the non-reducing end, attacks the adjacent α-1,4-linkage, and transfers it to the C-4 position of the non-reducing end to produce α-, β-, or γ-CD (Fig.5 A). This view was only confirmed from the analysis of CGTase action on 14C-labeled linear α-1,4-glucans with DP 7–12 (27Kobayashi S. Kainuma K. Suzuki S. Proceedings of Symposium of Amylases. 8. The Amylase Research Society of Japan, Osaka, Japan1973: 29-36Google Scholar), but not investigated in high molecular weight glucans. If this view can be applied to the CGTase action on high molecular weight glucans, cyclic products throughout the reaction on amylose are expected to be only α-, β-, and γ-CD. However, the results presented in this paper clearly demonstrate that the cyclic glucans produced in the initial stage of cyclization reaction of CGTase are not only α-, β-, and γ-CD, but are cyclic α-1,4-glucans with various DP ranging from 6 to more than 60. Large cyclic α-1,4-glucans were preferentially produced in the initial stage of cyclization reaction, which were subsequently converted into small cyclic α-1,4-glucans and into the final major products, α-CD or β-CD. Thus these findings apparently contradict the widely held view of the action model of CGTase, and so we propose a new model for the action of CGTase as shown in Fig. 5 B. CGTase probably attacks any α-1,4-linkage within the amylose molecule, and then transfers the newly formed reducing end of the substrate either to the non-reducing end of a separate linear acceptor molecule or glucose (the intermolecular transglycosylation or disproportionation reaction), or to its own non-reducing end (the intramolecular transglycosylation or cyclization reaction, Fig. 5 B). This random cyclization reaction produces wide ranges of cyclic α-1,4-glucans with DP 6 to more than 60. The reversibility of these reactions allows large cyclic molecules to be linearized again by transglycosylation, and smaller cyclic molecules to be subsequently produced. The equilibrium of the whole reaction tends toward the formation of α- or β-CD as the final major products. Both CGTase and D-enzyme catalyze the cyclization and disproportionation of α-1,4-glucan and transglycosidic linearization of cyclic α-1,4-glucan in the presence of a suitable acceptor molecule. During the cyclization reaction, large cyclic α-1,4-glucans were preferentially produced in the initial stage, but were subsequently converted into smaller cyclic α-1,4-glucans in both cases. Thus both enzymes seem to catalyze the same reaction, with the major difference being in the smallest size of the cyclic α-1,4-glucans produced. The DP of the smallest cyclic α-1,4-glucan produced by CGTase is 6. On the other hand, D-enzyme never produced α-, β-, and γ-CD and the smallest cyclic α-1,4-glucan has DP of 17. It is very interesting to know how the specificities of these products are determined differently between D-enzyme and CGTase. In our previous paper (23Takata H. Takaha T. Okada S. Takagi M. Imanaka T. J. Bacteriol. 1996; 178: 1600-1606Crossref PubMed Google Scholar), we discussed that cyclic α-1,4-glucan with DP 6–8 (α-, β-, and γ-CD) and those with DP more than 17 may have different structures. The structures of cyclic α-1,4-glucan with DP 6–8 (α-, β-, and γ-CD) may fit well to the active site of CGTase, and those with DP more than 17 may fit well to D-enzyme. However, this idea seems to be unlikely because it is now understood that CGTase can also produce cyclic α-1,4-glucans with DP more than 17. One possible explanation for the different product specificity is as follows. The equilibrium of the reaction catalyzed by both enzymes tends toward the formation of smaller cyclic α-1,4-glucans; however, the smallest cyclic α-1,4-glucan molecule to be produced by D-enzyme has DP of 17, whereas that of CGTase is 6. At the moment, we do not know the mechanism to determine the smallest cyclic product of each enzyme, although we speculate that the difference in the smallest products of each enzyme is attributable to the active site structure. Despite the similarity found in their activities, D-enzyme and CGTase show no similarity in their primary sequences (22Takaha T. Yanase M. Okada S. Smith S.M. J. Biol. Chem. 1993; 268: 1391-1396Abstract Full Text PDF PubMed Google Scholar). The tertiary structure of CGTase has already been obtained by x-ray crystallographic studies (3Klein C. Schulz G.E. J. Mol. Biol. 1991; 217: 737-750Crossref PubMed Scopus (214) Google Scholar, 4Knegtel R.M.A. Wind R.D. Rozeboom H.J. Kalk K.H. Buitelaar R.M. Dijkhuizen L. Dijkstra B.W. J. Mol. Biol. 1996; 256: 611-622Crossref PubMed Scopus (85) Google Scholar, 5Kubota M. Matsuura Y. Sakai S. Katsube Y. Oyo Toshitsu Kagaku. 1994; 41: 245-253Google Scholar, 6Lawson C.L. van Montfort R. Strokopytov B. Rozeboom H.J. Kalk K.H. de Vries G.E. Penninga D. Dijkhuizen L. Dijkstra B.W. J. Mol. Biol. 1994; 236: 590-600Crossref PubMed Scopus (221) Google Scholar). Determination of the structure of D-enzyme and its comparison with CGTase will be necessary to answer this question. We especially thank Shimadzu Co. for TOF-MS analyses." @default.
• W2091551142 created "2016-06-24" @default.
• W2091551142 creator A5006864913 @default.
• W2091551142 creator A5023390357 @default.
• W2091551142 creator A5057622558 @default.
• W2091551142 creator A5061201864 @default.
• W2091551142 creator A5062044136 @default.
• W2091551142 date "1997-06-01" @default.
• W2091551142 modified "2023-10-13" @default.
• W2091551142 title "Cyclodextrins Are Not the Major Cyclic α-1,4-Glucans Produced by the Initial Action of Cyclodextrin Glucanotransferase on Amylose" @default.
• W2091551142 cites W1526272699 @default.
• W2091551142 cites W1601651086 @default.
• W2091551142 cites W1918349277 @default.
• W2091551142 cites W1925779720 @default.
• W2091551142 cites W1975700344 @default.
• W2091551142 cites W1980646456 @default.
• W2091551142 cites W1986071039 @default.
• W2091551142 cites W1993278548 @default.
• W2091551142 cites W2007016282 @default.
• W2091551142 cites W2015055160 @default.
• W2091551142 cites W2016182682 @default.
• W2091551142 cites W2016301662 @default.
• W2091551142 cites W2019429599 @default.
• W2091551142 cites W2043534078 @default.
• W2091551142 cites W2052344553 @default.
• W2091551142 cites W2054064012 @default.
• W2091551142 cites W2054624750 @default.
• W2091551142 cites W2073154503 @default.
• W2091551142 cites W2076805087 @default.
• W2091551142 cites W2103929385 @default.
• W2091551142 cites W2110785282 @default.
• W2091551142 cites W2124358213 @default.
• W2091551142 cites W2151152537 @default.
• W2091551142 cites W2158205192 @default.
• W2091551142 doi "https://doi.org/10.1074/jbc.272.25.15729" @default.
• W2091551142 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9188466" @default.
• W2091551142 hasPublicationYear "1997" @default.
• W2091551142 type Work @default.
• W2091551142 sameAs 2091551142 @default.
• W2091551142 citedByCount "130" @default.
• W2091551142 countsByYear W20915511422012 @default.
• W2091551142 countsByYear W20915511422013 @default.
• W2091551142 countsByYear W20915511422014 @default.
• W2091551142 countsByYear W20915511422015 @default.
• W2091551142 countsByYear W20915511422016 @default.
• W2091551142 countsByYear W20915511422017 @default.
• W2091551142 countsByYear W20915511422018 @default.
• W2091551142 countsByYear W20915511422019 @default.
• W2091551142 countsByYear W20915511422020 @default.
• W2091551142 countsByYear W20915511422021 @default.
• W2091551142 countsByYear W20915511422022 @default.
• W2091551142 countsByYear W20915511422023 @default.
• W2091551142 crossrefType "journal-article" @default.
• W2091551142 hasAuthorship W2091551142A5006864913 @default.
• W2091551142 hasAuthorship W2091551142A5023390357 @default.
• W2091551142 hasAuthorship W2091551142A5057622558 @default.
• W2091551142 hasAuthorship W2091551142A5061201864 @default.
• W2091551142 hasAuthorship W2091551142A5062044136 @default.
• W2091551142 hasBestOaLocation W20915511421 @default.
• W2091551142 hasConcept C121332964 @default.
• W2091551142 hasConcept C178790620 @default.
• W2091551142 hasConcept C185592680 @default.
• W2091551142 hasConcept C2776804113 @default.
• W2091551142 hasConcept C2779433975 @default.
• W2091551142 hasConcept C2780791683 @default.
• W2091551142 hasConcept C529335014 @default.
• W2091551142 hasConcept C62520636 @default.
• W2091551142 hasConceptScore W2091551142C121332964 @default.
• W2091551142 hasConceptScore W2091551142C178790620 @default.
• W2091551142 hasConceptScore W2091551142C185592680 @default.
• W2091551142 hasConceptScore W2091551142C2776804113 @default.
• W2091551142 hasConceptScore W2091551142C2779433975 @default.
• W2091551142 hasConceptScore W2091551142C2780791683 @default.
• W2091551142 hasConceptScore W2091551142C529335014 @default.
• W2091551142 hasConceptScore W2091551142C62520636 @default.
• W2091551142 hasIssue "25" @default.
• W2091551142 hasLocation W20915511421 @default.
• W2091551142 hasOpenAccess W2091551142 @default.
• W2091551142 hasPrimaryLocation W20915511421 @default.
• W2091551142 hasRelatedWork W1531601525 @default.
• W2091551142 hasRelatedWork W2319480705 @default.
• W2091551142 hasRelatedWork W2384464875 @default.
• W2091551142 hasRelatedWork W2606230654 @default.
• W2091551142 hasRelatedWork W2607424097 @default.
• W2091551142 hasRelatedWork W2748952813 @default.
• W2091551142 hasRelatedWork W2899084033 @default.
• W2091551142 hasRelatedWork W2948807893 @default.
• W2091551142 hasRelatedWork W4387497383 @default.
• W2091551142 hasRelatedWork W2778153218 @default.
• W2091551142 hasVolume "272" @default.
• W2091551142 isParatext "false" @default.
• W2091551142 isRetracted "false" @default.
• W2091551142 magId "2091551142" @default.
• W2091551142 workType "article" @default.