Characterization and structural analyses of a novel glycosyltransferase acting on the β-1,2-glucosidic linkages
2022; Elsevier BV; Volume: 298; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2022.101606
ISSN1083-351X
AutoresKaito Kobayashi, Hisaka Shimizu, Nobukiyo Tanaka, Kouji Kuramochi, Hiroyuki Nakai, Masahiro Nakajima, Hayao Taguchi,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoThe IALB_1185 protein, which is encoded in the gene cluster for endo-β-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are β-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward β-1,2-glucooligosaccharides, and that the kinetic parameters for β-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the β-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites −1 and +1, whereas in the E343Q mutant–sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites −1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of β-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined β-1,2-glucooligosaccharide:d-glucoside β-d-glucosyltransferase as a systematic name and β-1,2-glucosyltransferase as an accepted name. The IALB_1185 protein, which is encoded in the gene cluster for endo-β-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are β-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward β-1,2-glucooligosaccharides, and that the kinetic parameters for β-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the β-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites −1 and +1, whereas in the E343Q mutant–sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites −1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of β-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined β-1,2-glucooligosaccharide:d-glucoside β-d-glucosyltransferase as a systematic name and β-1,2-glucosyltransferase as an accepted name. Carbohydrate chains are important polymer compounds for all organisms, which is attributed to the wide variety of carbohydrate chain structures. Such complexity of the structures is thought to be responsible for the repertoire of enzymes that synthesize and degrade carbohydrate chains. The functions and structures of these enzymes have become extensively diversified through molecular evolution. To date, various kinds of enzymes related to carbohydrates have been found and added to the Carbohydrate-Active enZYmes (CAZy) database (http://www.cazy.org) (1Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42: 490-495Google Scholar, 2Henrissat B. Vegetales M. Grenoble F. A classification of glycosyl hydrolases based sequence similarities amino acid.Biochem. J. 1991; 280: 309-316Google Scholar). 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Bacteriol. 1994; 176: 6538-6544Google Scholar). Glycosides possessing β-1,2-glucooligosaccharides (Sopns, n is DP) as glycones have been found in plants such as stevia, citrus fruits, and so on (15Brahmachari G. Mandal L.C. Roy R. Sadhan M. Brahmachari A.K. Stevioside and related compounds – molecules of pharmaceutical promise: A critical overview.Arch. Pharm. Chem. Life Sci. 2011; 344: 5-19Google Scholar, 16Hillebrand S. Schwarz M. Winterhalter P. Characterization of anthocyanins and pyranoanthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juice.J. Agric. Food Chem. 2004; 52: 7331-7338Google Scholar). Sophorolipids are found in many yeast species (17Claus S. Van Bogaert I.N.A. Sophorolipid production by yeasts: A critical review of the literature and suggestions for future research.Appl. Microbiol. Biotechnol. 2017; 101: 7811-7821Google Scholar), and ones from Candida bombicola are commercially manufactured (18Felse P.A. Shah V. Chan J. Rao K.J. Gross R.A. 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However, the physical and physiological functions of β-1,2-glucans have not been investigated sufficiently, which might be related to less progress in exploration of β-1,2-glucan-associated enzymes. Genes encoding β-1,2-glucan-degrading enzymes had not been identified until a novel phosphorylase was first found in Listeria innocua as an enzyme that can act on linear β-1,2-glucans (Hereafter, β-1,2-glucan represents a linear form unless otherwise noted.) in 2014 (20Kitahata S. Edagawa S. Cyclic (1→2)-β-D-glucan-hydrolyzing enzymes from Acremonium sp. 15: Purification and some properties of endo-(1→2)-β-D-glucanase and β-D-glucosidase.Agric. Biol. Chem. 1987; 51: 2701-2708Google Scholar, 21Mendoza N.S. Amemura A. (1,2)-β-D-glucan-hydrolyzing enzymes in Cytophaga arvensicola: Partial purification and some properties of endo-(1,2)-β-glucanase and β-D-glucosidase specific for (1,2)- and (1,3)-linkages.J. Ferment. Technol. 1983; 61: 473-481Google Scholar, 22Reese E.T. Parrish F.W. Mandels M. β-D-1,2-Glucanases in fungi.Can. J. Microbiol. 1961; 7: 309-317Google Scholar). This enzyme was named 1,2-β-oligoglucan phosphorylase (SOGP) and was given a new EC number (EC2.4.1.333) (23Nakajima M. Toyoizumi H. Abe K. Nakai H. Taguchi H. Kitaoka M. 1,2-β-Oligoglucan phosphorylase from Listeria innocua.PLoS One. 2014; 9e92353Google Scholar). After that, a putative glycoside hydrolase family (GH) 3 enzyme in the SOGP gene cluster was found to be a β-glucosidase preferably hydrolyzing Sop2 by functional and structural analyses (24Nakajima M. Yoshida R. Miyanaga A. Abe K. Takahashi Y. Sugimoto N. Toyoizumi H. Nakai H. Kitaoka M. Taguchi H. Functional and structural analysis of a β-glucosidase involved in β-1,2-glucan metabolism in Listeria innocua.PLoS One. 2016; 11e0148870Google Scholar). A carbohydrate-binding subunit of a putative ABC transporter in the same gene cluster was also found to be a Sopns-binding protein (25Abe K. Sunagawa N. Terada T. Takahashi Y. Arakawa T. Igarashi K. Samejima M. Nakai H. Taguchi H. Nakajima M. Fushinobu S. Structural and thermodynamic insights into β-1,2-glucooligosaccharide capture by a solute-binding protein in Listeria innocua.J. Biol. Chem. 2018; 293: 8812-8828Google Scholar). These results are the first biochemical evidence of the existence of a gene cluster involved in β-1,2-glucan metabolism. A large-scale preparation method for β-1,2-glucan has been established using SOGP and inexpensive sugars as materials (26Abe K. Nakajima M. Kitaoka M. Toyoizumi H. Takahashi Y. Sugimoto N. Nakai H. Taguchi H. Large-scale preparation of 1,2-β-glucan using 1,2-β-oligoglucan phosphorylase.J. Appl. Glycosci. 2015; 62: 47-52Google Scholar, 27Kobayashi K. Nakajima M. Aramasa H. Kimura S. Iwata T. Nakai H. Taguchi H. Large-scale preparation of β-1,2-glucan using quite a small amount of sophorose.Biosci. Biotechnol. Biochem. 2019; 83: 1867-1874Google Scholar). The prepared β-1,2-glucan was used for identification of endo-β-1,2-glucanases (SGLs) from a bacterium and a fungus. Both SGLs have been successfully identified and classified into new families (GH144 and GH162, respectively) (28Abe K. Nakajima M. Yamashita T. Matsunaga H. Kamisuki S. Nihira T. Takahashi Y. Sugimoto N. Miyanaga A. Nakai H. Arakawa T. Fushinobu S. Taguchi H. Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family.J. Biol. Chem. 2017; 292: 7487-7506Google Scholar, 29Tanaka N. Nakajima M. Narukawa-Nara M. Matsunaga H. Kamisuki S. Aramasa H. Takahashi Y. Sugimoto N. Abe K. Terada T. Miyanaga A. Yamashita T. Sugawara F. Kamakura T. Komba S. et al.Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family.J. Biol. Chem. 2019; 294: 7942-7965Google Scholar). This finding enables us to explore SGL homologs and SGL gene clusters. An SGL homolog possessing an unknown function region at the N terminus has been identified as a novel exolytic enzyme that releases Sop2 from the nonreducing ends of Sopns (30Shimizu H. Nakajima M. Miyanaga A. Takahashi Y. Tanaka N. Kobayashi K. Sugimoto N. Nakai H. Taguchi H. Characterization and structural analysis of a novel exo-type enzyme acting on β-1,2-glucooligosaccharides from Parabacteroides distasonis.Biochemistry. 2018; 57: 3849-3860Google Scholar). A β-glucosidase preferably acting on longer Sopns and β-1,2-glucan has also been found from an SGL gene cluster in Bacteroides thetaiotaomicron (31Ishiguro R. Tanaka N. Abe K. Nakajima M. Maeda T. Miyanaga A. Takahashi Y. Sugimoto N. Nakai H. Taguchi H. Function and structure relationships of a β-1,2-glucooligosaccharide-degrading β-glucosidase.FEBS Lett. 2017; 591: 3926-3936Google Scholar). The structure–function relationships of these enzymes have also been analyzed (24Nakajima M. Yoshida R. Miyanaga A. Abe K. Takahashi Y. Sugimoto N. Toyoizumi H. Nakai H. Kitaoka M. Taguchi H. Functional and structural analysis of a β-glucosidase involved in β-1,2-glucan metabolism in Listeria innocua.PLoS One. 2016; 11e0148870Google Scholar, 25Abe K. Sunagawa N. Terada T. Takahashi Y. Arakawa T. Igarashi K. Samejima M. Nakai H. Taguchi H. Nakajima M. Fushinobu S. Structural and thermodynamic insights into β-1,2-glucooligosaccharide capture by a solute-binding protein in Listeria innocua.J. Biol. Chem. 2018; 293: 8812-8828Google Scholar, 28Abe K. Nakajima M. Yamashita T. Matsunaga H. Kamisuki S. Nihira T. Takahashi Y. Sugimoto N. Miyanaga A. Nakai H. Arakawa T. Fushinobu S. Taguchi H. Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family.J. Biol. Chem. 2017; 292: 7487-7506Google Scholar, 29Tanaka N. Nakajima M. Narukawa-Nara M. Matsunaga H. Kamisuki S. Aramasa H. Takahashi Y. Sugimoto N. Abe K. Terada T. Miyanaga A. Yamashita T. Sugawara F. Kamakura T. Komba S. et al.Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family.J. Biol. Chem. 2019; 294: 7942-7965Google Scholar, 32Nakajima M. Tanaka N. Furukawa N. Nihira T. Kodutsumi Y. Takahashi Y. Sugimoto N. Miyanaga A. Fushinobu S. Taguchi H. Nakai H. Mechanistic insight into the substrate specificity of 1,2-β-oligoglucan phosphorylase from Lachnoclostridium phytofermentans.Sci. Rep. 2017; 7: 42671Google Scholar). However, the abovementioned reports are most of the studies on β-1,2-glucan-degrading enzymes, implying insufficient understanding of the variety of β-1,2-glucan-associated enzymes. Here, we focus on an SGL gene cluster in the genome from Ignavibacterium album, a moderately thermophilic anaerobic bacterium found in a hot spring in Japan (33Iino T. Mori K. Uchino Y. Nakagawa T. Harayama S. Suzuki K.I. Ignavibacterium album gen. nov., sp. nov., a moderately thermophilic anaerobic bacterium isolated from microbial mats at a terrestrial hot spring and proposal of Ignavibacteria classis nov., for a novel lineage at the periphery of green sulfur bacteria.Int. J. Syst. Evol. Microbiol. 2010; 60: 1376-1382Google Scholar). This gene cluster includes two genes encoding putative GH144 enzymes, and β-1,2-glucan-related genes encoding a putative GH3 β-glucosidase, a putative GH94 enzyme (a homolog of the SOGP), and a putative Sopns-binding protein in an ABC transporter (Fig. S1). The gene cluster also contains a gene (ialb_1185) encoding a putative GH35 enzyme (IALB_1185, hereafter IaSGT). While GH35 enzymes, which are distributed in a wide range of microorganisms, plants, and animals, are mainly β-galactosidases (β-galactosidase, exo-β-1,4-galactanase, and β-1,3-galactosidase) according to the CAZy database (34Cheng W. Wang L. Jiang Y.L. Bai X.H. Chu J. Li Q. Yu G. Liang Q.L. Zhou C.Z. Chen Y. Structural insights into the substrate specificity of Streptococcus pneumoniae β(1,3)-galactosidase BgaC.J. Biol. Chem. 2012; 287: 22910-22918Google Scholar, 35Goulas T. Goulas A. Tzortzis G. Gibson G.R. Comparative analysis of four β-galactosidases from Bifidobacterium bifidum NCIMB41171: Purification and biochemical characterisation.Appl. Microbiol. Biotechnol. 2009; 82: 1079-1088Google Scholar, 36Kondo T. Nishimura Y. Matsuyama K. Ishimaru M. Nakazawa M. Ueda M. Sakamoto T. Characterization of three GH35 β-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.Appl. Microbiol. Biotechnol. 2020; 104: 1135-1148Google Scholar), several GH35 enzymes from Archaea have been found to be β-glucosaminindases (GlmAs), and GlmA from Thermococcus kodakaraensis (TkGlmA) hydrolyzes chitosan and chitooligosaccharides (37Liu B. Li Z. Hong Y. Ni J. Sheng D. Shen Y. Cloning, expression and characterization of a thermostable exo-β-D-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii.Biotechnol. Lett. 2006; 28: 1655-1660Google Scholar, 38Tanaka T. Fukui T. Atomi H. Imanaka T. Characterization of an exo-β-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.J. Bacteriol. 2003; 185: 5175-5181Google Scholar). The binding modes of natural substrates are not sufficiently understood, since only the glucosamine (GlcN) complex is available among GlmAs (39Mine S. Watanabe M. Structural insights into the molecular evolution of the archaeal exo-β-D-glucosaminidase.Int. J. Mol. Sci. 2019; 20: 2460Google Scholar). Though many GH35 enzymes have been reported, as described above, no glucoside-acting enzyme has been reported in this family. In this study, we report the first β-1,2-glucan-associated GH35 enzyme biochemically and structurally and furthermore describe why the enzyme is a novel enzyme that should be given a new EC number. Phylogenetic analysis was performed using the amino acid sequences of characterized GH35 enzymes in the CAZy database and IaSGT. While Eukaryotic GH35 enzymes are divided into two clusters, each bacterial and archaeal GH35 enzymes form one cluster, respectively (Fig. S2 and Table S1). Notably, IaSGT and its homologs form a distinct cluster from the known GH35 enzymes. Though the group of archaeal GlmAs is close to that of IaSGT in the phylogenetic tree, the amino acid sequence identity between these enzymes and IaSGT is low (only ∼28%). IaSGT has no N-terminal signal peptide, suggesting that it is localized in the cytosol. Nucleophile and acid/base residues in GlmAs (Glu347 and Glu179 in TkGlmA, respectively) are conserved in IaSGT (E343 and E176, respectively) (Fig. S3). Though most substrate recognition residues at subsite −1 in the GlmAs (Tyr53, Glu103, Glu179, Glu347, and Tyr379 in TkGlmA) are also conserved in IaSGT, aspartate residues (Asp178 in TkGlmA) considered to be responsible for specificity to the amino group in GlcN are replaced by an asparagine residue in IaSGT (Asn175). Furthermore, subsite plus side regions (around Glu184 and Leu282 in TkGlmA) are not conserved at all. These differences imply that IaSGT has different substrate specificity from the GlmAs. The purified IaSGT migrated as a single band corresponding to approximately 75 kDa/m on an SDS-PAGE gel (Fig. S4A). The enzyme was eluted at the time corresponding to 141 kDa/m on size-exclusion chromatography (Fig. S4B). Thus, this enzyme should form a dimer. Since IaSGT acted on Sop2 to release glucose (Glc) as described below in detail, quantification of Glc was used for investigation of pH and temperature profiles. IaSGT showed high activity at pH 5.0 − 8.0 (over 90% relative activity as to the highest) and was stable at pH 5.0 to 11.0 (Fig. S5A). IaSGT showed optimum activity at 55 °C and was stable up to 60 °C after incubation for 1 h (Fig. S5B), which is consistent with the bacterial growth property as to temperature. Since most GH35 enzymes show β-galactosidase activity, the activity of IaSGT toward β-galactosides was investigated. However, IaSGT did not show any hydrolytic activity toward lactose (β-Gal-1,4-Glc) (Fig. 1A) or p-nitrophenyl (pNP)-β-galactopyranoside (Gal), an artificial substrate (less than 0.01 U/mg for 1 mM pNP-β-Gal). Nor did the enzyme act on various oligosaccharides such as cellooligosaccharides (Cel2−5), laminarioligosaccharides (Lam2−5), maltose (α-Glc-1,4-Glc), gentiobiose (β-Glc-1,6-Glc), or sucrose (α-Glc-1,2-β-Fru) (Fig. 1, A–C). On the other hand, IaSGT showed activity toward Sop2−5 obviously (Fig. 1D). IaSGT produced oligosaccharides with both lower and higher DPs than those of the substrates. This disproportionation of DPs proceeded by transfer of a glucose unit. The products appeared not to show decreases in their average DPs even after reaction overnight. The enzyme did not show hydrolytic (glucose-releasing) activity toward Sop3 (less than 0.01 U/mg for 20 mM Sop3). These results indicated that the reaction mode of IaSGT was that of a glycosyltransferase. After the products at the beginning of the reaction with Sop3 were fractionated by size-exclusion chromatography, only the product at the position corresponding to Sop4 on the TLC plate was collected and analyzed by 1H-NMR (Fig. S6A). The chemical shifts of the product fitted completely with those of the reference Sop4 (Fig. S6, B and C), indicating that the reaction product is Sop4 and that the enzyme transfers a glucose unit to produce a β-1,2-glucosidic bond. Such elongation by transfer of Glc units has been found in GH16 elongating β-transglycosylase, though the GH16 enzyme acts on β-1,3/1,4-linkages (40Qin Z. Yang S. Zhao L. You X. Yan Q. Jiang Z. Catalytic mechanism of a novel glycoside hydrolase family 16 "elongating" β-transglycosylase.J. Biol. Chem. 2017; 292: 1666-1678Google Scholar). To understand the DPs of the reaction products in detail, the products after the overnight reaction with Sop5 were separated on the TLC plate by developing twice. As a result, Sopns with DP at least up to 9 were clearly detected (Fig. 1E). This is consistent with the fact that Sop2−9 were clearly detected by electrospray ionization–mass spectrometry (ESI-MS) (Fig. S7A). Sopns with DPs of 10 or more could also be assigned. Though velocities of disproportionation appeared to slow down after 3 h of the reactions (Fig. 1D), this is probably because the proportion of Sopns molecules with the highest and the lowest DPs to all the substrate molecules in the reaction solution was reduced. In order to determine whether Sopns are appropriate substrates for IaSGT, the kinetic parameters of the glucosyl transferase activity of the enzyme toward Sop2−5 were determined (Table 1). Though IaSGT showed modest kcat values, the Km values were remarkably large, especially for Sop2 and Sop3 (120 mM and 300 mM, respectively), as a GH enzyme. Consequently, the kcat/Km values were quite small (less than 0.1 s−1 mM−1 for Sop2, Sop3, and Sop5, and less than 0.5 s−1 mM−1 for Sop4). Since the substrates in the transferase reaction are both donors and acceptors, the quite large Km values suggest that Sopns are inappropriate as at least either donors or acceptors.Table 1Kinetic parameters of IaSGT for SopnsSubstratekcat (s−1)Km (mM)kcat/Km (s−1 mM−1)Sop28.0 ± 0.9120 ± 200.069 ± 0.002Sop318 ± 1300 ± 200.059 ± 0.001Sop44.8 ± 0.215 ± 20.32 ± 0.02Sop51.7 ± 0.222 ± 30.078 ± 0.005Concentrations used were 5 to 40 mM, except for Sop3 (5–80 mM). All experiments were carried out in triplicate. Standard errors are used in the table. Open table in a new tab Concentrations used were 5 to 40 mM, except for Sop3 (5–80 mM). All experiments were carried out in triplicate. Standard errors are used in the table. To determine optimal acceptors of IaSGT, the effects of various monosaccharides and disaccharides (1 mM d-mannose, d-glucose, d-galactose, d-xylose, d-talose, l-arabinose, d-fructose, l-rhamnose, d-gluconate, Lam2, Cel2, gentiobiose, sucrose, maltose, α,α-trehalose, or lactose) as acceptors on activity toward 2 mM Sop2 were investigated. However, a remarkable increase in specific activity was not found (less than 15% increase in specific activity, data not shown). Then, the glycosynthase activity of the E343G mutant in the presence of α-d-glucosyl fluoride (α-GlcF) as a donor was investigated by TLC analysis. The mutant showed glycosynthase activity only in the presence of glucose as an acceptor among the examined monosaccharides and disaccharides, though α-GlcF itself acted as an acceptor as well (Fig. S8). When pNP-α-Glc was used as an acceptor, a synthetic product was observed. Therefore, various aryl- and alkyl-glucosides, as acceptors, were investigated using the WT IaSGT in the presence of Sop2 as a donor. Reaction products were detected regardless of the anomer of acceptors except methyl-β-Glc (Fig. 2). Considering the kinetic analysis described later, spots of reaction products derived from methyl-β-Glc seemed to overlap those of Sopns. ESI-MS analysis using phenyl-α-Glc and Sop2 detected peaks assigned as the compounds of phenyl-α-Glc linked with one or two Glc units clearly, though the peak corresponding to Glc was small (Fig. S7B). This result is consistent with detection of two spots indicated by arrows below the spot of phenyl-α-Glc in the TLC plate (Fig. 2). We determined the kinetic parameters of the glycosyltransfer activity of IaSGT using Sop2 as a donor and various glucosides as acceptors. The enzyme showed remarkably higher activity toward most of the investigated acceptors than that in the absence of the acceptors (Table 2). The Km values for the α-glucosides were at the range of 0.044−0.38 mM (approximately 300−2600 times smaller than that of Sop2 without the acceptors), and the kcat/Km values for the acceptors were approximately 70−570 times higher than that of Sop2 without an acceptor. In the case of β-glucosides, the Km values were in the range of 0.021−0.15 mM (approximately 770−5500 times smaller than that of Sop2 without the acceptors), and kcat/Km values were approximately 30−800 times higher than that of Sop2 without an acceptor. Overall, β-glucosides showed smaller Km and kcat values than those of α-glucosides. However, the kcat/Km values for acceptors with both types of anomers were within a similar range and were sufficiently large as those of GH enzymes. Therefore, the enzyme can act on a wide range of glucosides with various aryl- and alkyl-groups and both types of anomers as acceptors. In addition, IaSGT comparably acted on amygdalin, a gentiobioside, as an acceptor, though the Km and kcat/Km values were rather larger and smaller than those of the β-glucosides, respectively.Table 2Kinetic parameters of IaSGT for acceptors and donorsSubstratekcat (s−1)Km (mM)kcat/Km (s−1 mM−1)Acceptora0.025 to 0.4 mM acceptors were used. A fixed concentr
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