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A UDP-Glucose:Isoflavone 7-O-Glucosyltransferase from the Roots of Soybean (Glycine max) Seedlings

2007; Elsevier BV; Volume: 282; Issue: 32 Linguagem: Inglês

10.1074/jbc.m702651200

1083-351X

Akio Noguchi, Atsushi Saito, Yu Homma, Masahiro Nakao, Nobuhiro Sasaki, Tokuzo Nishino, Seiji Takahashi, Tôru Nakayama,

Plant-Microbe Interactions and Immunity

Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-β-d-glucosides (kcat = 0.74 s-1, Km for genistein = 3.6 μm, and Km for UDP-glucose = 190 μm). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1. Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-β-d-glucosides (kcat = 0.74 s-1, Km for genistein = 3.6 μm, and Km for UDP-glucose = 190 μm). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1. Isoflavones are a class of plant flavonoids with a 3-phenylchromone structure and occur predominantly in legumes, where these flavonoids play very important roles in plant-microbe interactions. For example, the isoflavone genistein is excreted from soybean (Glycine max L. Merr.) roots to serve as a signaling molecule in rhizobia-mediated nodulation of this plant (1Phillips D. Stafford H. Ibrahim R. Recent Adv. Phytochem. 1992; 26: 201-231Google Scholar, 2Hungria M. Stacey G. Soil Biol. Biochem. 1997; 29: 819-830Crossref Scopus (111) Google Scholar). They are also involved in defensive mechanisms of legumes against pathogen infection (3Barz W. Welle R. Stafford H. Ibrahim R. Phenolic Metabolism in Plants. 26. 1992: 139-164Google Scholar, 4Graham T. Graham M. Stacey G. Keen N.T. Plant-Microbe Interactions. 5. 2000: 181-220Google Scholar). Moreover, isoflavones exhibit a wide variety of bioactivities that are beneficial to human health (5Wiseman H. Andersen Ø.M. Markham K.R. Flavonoids: Chemistry, Biochemistry and Applications. 2006: 371-396Google Scholar). The isoflavonoid skeleton is derived from (2S)-flavanones, a class of general flavonoid intermediates that undergo 2-hydroxylation catalyzed by 2-hydroxyflavanone synthase, a microsomal cytochrome P450 enzyme (see Fig. 1) (6Akashi T. Aoki T. Ayabe S. Plant Physiol. 1999; 121: 821-828Crossref PubMed Scopus (174) Google Scholar, 7Steele C.L. Gijzen M. Qutob D. Dixon R.A. Arch. Biochem. Biophys. 1999; 367: 146-150Crossref PubMed Scopus (168) Google Scholar). In G. max and some other legumes, the resulting product, 2,5,7,4′-tetrahydroxyisoflavanone or 2,7,4′-trihydroxyisoflavanone, then undergoes enzymatic dehydration to produce genistein or daidzein, respectively (8Akashi T. Aoki T. Ayabe S. Plant Physiol. 2005; 137: 882-891Crossref PubMed Scopus (116) Google Scholar). The resulting isoflavone aglycons are 7-O-glucosylated and subsequently 6″-O-malonylated (9Suzuki H. Nishino T. Nakayama T. Phytochemistry. 2007; 10.1016/j.phytochem.2007.05.017Google Scholar) to produce isoflavone conjugates, which then accumulate in vacuoles (3Barz W. Welle R. Stafford H. Ibrahim R. Phenolic Metabolism in Plants. 26. 1992: 139-164Google Scholar). The release of aglycons from these conjugates is catalyzed by an isoflavone conjugate-hydrolyzing β-glucosidase. We showed previously that, in G. max seedlings, this β-glucosidase is localized exclusively in the root apoplast (10Suzuki H. Takahashi S. Watanabe R. Fukushima Y. Fujita N. Noguchi A. Yokoyama R. Nishitani K. Nishino T. Nakayama T. J. Biol. Chem. 2006; 281: 30251-30259Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Most biological and biomedical activities of isoflavones have been identified with their aglycon forms (4Graham T. Graham M. Stacey G. Keen N.T. Plant-Microbe Interactions. 5. 2000: 181-220Google Scholar, 5Wiseman H. Andersen Ø.M. Markham K.R. Flavonoids: Chemistry, Biochemistry and Applications. 2006: 371-396Google Scholar, 11Pueppke S.G. Bolanos-Vasquez M.C. Werner D. Bec-Ferte M.P. Prome J.C. Krishnan H.B. Plant Physiol. 1998; 117: 599-606Crossref PubMed Scopus (61) Google Scholar), and conjugation can affect the pharmacokinetics of the dietary isoflavonoids (5Wiseman H. Andersen Ø.M. Markham K.R. Flavonoids: Chemistry, Biochemistry and Applications. 2006: 371-396Google Scholar). Therefore, the conjugation of isoflavones is important for controlling the interactions of legumes with their symbiotic and pathogenic microorganisms as well as the dietary effects of these flavonoids on human health. The 7-O-glucosylation is the first step of isoflavone conjugation and is specifically catalyzed by G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) 2The abbreviations used are: GmIF7GT, G. max UDP-glucose:isoflavone 7-O-glucosyltransferase; PSPGs, plant secondary product glycosyltransferases; VvGT1, V. vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase-1; IFGT, UDP-glucose:isoflavone glucosyltransferase; HPLC, high performance liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RACE, rapid amplification cDNA ends; AmC4′GT, A. majus UDP-glucose:chalcone 4′-O-glucosyltransferase; LvC4′GT, L. vulgaris UDP-glucose:chalcone 4′-O-glucosyltransferase; SbB7GAT, S. baicalensis UDP-glucuronate:baicalein 7-O-glucuronosyltransferase; GeIF7GT, G. echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. (Fig. 1) (3Barz W. Welle R. Stafford H. Ibrahim R. Phenolic Metabolism in Plants. 26. 1992: 139-164Google Scholar). However, the primary structure and phylogenetics of this important glycosyltransferase of G. max have long remained unclear, mainly because of the difficulty of enzyme purification. Glycosyltransferases generally catalyze the transfer of the glycosyl group from nucleotide diphosphate-activated sugars to acceptor molecules. A vast variety of glycosyltransferase genes have been identified thus far, which are currently classified on the basis of their phylogenetics into >70 families (12Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (630) Google Scholar, 13Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar). So far, glycosyltransferases involved in plant secondary metabolism (i.e. plant secondary product glycosyltransferases (PSPGs)) have all been grouped into glycosyltransferase family 1 (14Vogt T. Jones P. Trends Plant Sci. 2000; 5: 380-386Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 15Keegstra K. Raikhel N. Curr. Opin. Plant Biol. 2001; 4: 219-224Crossref PubMed Scopus (123) Google Scholar, 16Vogt T. Jones P. Planta. 2001; 213: 164-174Crossref PubMed Scopus (329) Google Scholar, 17Lim E-K. Bowles D.J. EMBO J. 2004; 23: 2915-2922Crossref PubMed Scopus (193) Google Scholar, 18Bowles D.J. Isayenkova J. Lim E-K. Poppenberger B. Curr. Opin. Plant Biol. 2005; 8: 254-263Crossref PubMed Scopus (374) Google Scholar, 19Sawada S. Suzuki H. Ichimaida F. Yamaguchi M.A. Iwashita T. Fukui Y. Hemmi H. Nishino T. Nakayama T. J. Biol. Chem. 2005; 280: 899-906Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). These PSPGs are characterized by a unique well conserved sequence of ∼45 amino acid residues (called a PSPG box (14Vogt T. Jones P. Trends Plant Sci. 2000; 5: 380-386Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar)) and a catalytic mechanism leading to an inversion of the anomeric configuration of transferred sugar (12Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (630) Google Scholar). Recent crystallographic studies of PSPGs from Medicago truncatula (UGT71G1) (20Shao H. He X.Z. Achnine L. Blount J.W. Dixon R.A. Wang X. Plant Cell. 2005; 17: 3141-3154Crossref PubMed Scopus (299) Google Scholar) and Vitis vinifera (VvGT1) (21Offen W. Martinez-Fleites C. Yang M. Lim E-K. Davis B.G Tarling C.A Ford C.M Bowles D.J Davies G.J. EMBO J. 2006; 25: 1396-1405Crossref PubMed Scopus (358) Google Scholar), along with the results of site-directed mutagenesis studies of these enzymes (22He X.Z. Wang X. Dixon R.A. J. Biol. Chem. 2006; 281: 34441-34447Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), afforded important information relative to the mechanism and specificity of PSPG-catalyzed reactions. In both of these enzymes, a histidine residue (His-22 in UGT71G1 and His-20 in VvGT1) that is highly conserved among PSPGs is proposed to act as a key catalytic residue that activates the hydroxy group of the glucosyl acceptor molecule to facilitate glucosidic linkage formation. A well conserved aspartic acid residue (Asp-121 in UGT71G1 and Asp-119 in VvGT1) is hydrogen-bonded with the His residue and is proposed to assist in its general acid/base role during catalysis. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. This permitted the identification of the GmIF7GT cDNA and its phylogenetic analysis. The purified enzyme, which displayed a high glucosyl transfer activity, unexpectedly lacked an N-terminal 49-residue segment and thus was devoid of the conserved His residue (His-15 in GmIF7GT) that corresponds to the catalytically important residues His-22 in UGT71G1 and His-20 in VvGT1. This observation prompted the undertaking of the mutagenesis studies to evaluate the importance of His-15 as well as that of other conserved amino acid residues for GmIF7GT catalysis. Soybean seeds (G. max L. Merr. cv. Wase-Hakucho; Takii & Co., Ltd., Kyoto, Japan) were pretreated with running tap water for 10 min and then germinated on planting medium containing 10 mm potassium phosphate (pH 7.0), 1% (w/v) sucrose, and 0.5% (w/v) agar. The roots of 7-9-day-old seedlings were washed with tap water to remove the medium and frozen at -80 °C until used. Benzoic acid, m- and p-hydroxy-benzoic acids, salicylic acid, salicyl alcohol, hydroquinone, caffeic acid, trans-p-coumaric acid, and naringenin were purchased from Nacalai Tesque (Kyoto). Esculetin and esculin were from Tokyo Kasei Industries (Tokyo, Japan). Kaempferol and quercetin were obtained from Wako Pure Chemical Industries (Osaka, Japan). Genistein, genistin, daidzein, and daidzin were products of Fujicco Co., Ltd. (Kobe, Japan). UDP-glucose, UDP-galactose, and UDP-glucuronic acid were purchased from Sigma. All other chemicals were analytical grade. UDP-glucose:isoflavone glucosyltransferase (IFGT) activity was measured using genistein and UDP-glucose as substrates. The standard reaction mixture (100 μl) consisted of 200 μm genistein, 200 μm UDP-glucose, 50 mm Tris-HCl (pH 8.5), and enzyme. The mixture without enzyme was preincubated at 30 °C for 10 min, and the reaction was started by the addition of enzyme. After incubation at 30 °C for 10-30 min, the reaction was stopped by the addition of 100 μl of 0.5% (v/v) trifluoroacetic acid. The reaction products were analyzed by reversed-phase HPLC on a J’Sphere ODS-M80 column (4.6 × 150 mm; YMC, Kyoto). The substrates and products were eluted with a linear gradient of 13.5-90% (v/v) CH3CN containing 0.5% (v/v) trifluoroacetic acid in 8 min at a flow rate of 0.7 ml/min and detected at 260 nm using an SPD-10Avp UV-visible detector (Shimadzu, Kyoto). The protein concentration was determined by the Bradford method (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217529) Google Scholar) using bovine serum albumin as a standard. The concentration of purified GmIF7GT was determined by absorption coefficients ɛ280 = 46,410 m-1cm-1 (for the purified enzyme lacking an N-terminal 49-residue segment; see below) and 47,900 m-1cm-1 (for the full-length recombinant enzyme), which were calculated from the amino acid sequence. The initial velocity assays for GmIF7GT and its mutants were carried out under steady-state conditions using the standard assay system (see above) with varying concentrations of substrates. Apparent Km and Vmax values for glucosyl donor and acceptor substrates in the presence of a saturating concentration of their countersubstrate were determined by fitting the initial velocity data to a Michaelis-Menten equation by nonlinear regression analysis (24Leatherbarrow R.J. Trends Biochem. Sci. 1990; 15: 455-458Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 25Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. 1975; (, pp. , John Wiley & Sons, Inc., New York): 505-664Google Scholar). All operations were performed at 4 °C. All buffers used throughout the enzyme purification contained 56 mm 2-mercaptoethanol. Step 1: Preparation of Crude Extract—Frozen roots of G. max seedlings (1.2 kg) were suspended in 3800 ml of 0.1 m Tris-HCl (pH 8.5) containing 1 mm phenylmethylsulfonyl fluoride and 16% (w/v) polyvinylpolypyrrolidone and disrupted in a Waring blender, followed by centrifugation at 15,000 × g for 30 min. The supernatant was used for further purification. Step 2: Ammonium Sulfate Fractionation—The protein fraction that precipitated between 40 and 60% saturated ammonium sulfate was collected by centrifugation at 15,000 × g for 30 min. The pellet was dissolved in 800 ml of buffer S1 (20 mm Tris-HCl (pH 8.5)). Step 3: DEAE-Sepharose—The enzyme solution was applied to a DEAE-Sepharose Fast Flow column (1.6 × 51.5 cm; GE Healthcare) equilibrated with buffer S1 at a flow rate of 2 ml/min using anAöKTApurifier apparatus (GE Healthcare). The column was washed with the same buffer. The enzyme was eluted with a linear gradient of 0-0.36 m NaCl in 500 min in buffer S1. The active fractions were combined, concentrated, and equilibrated with buffer S1 by repeated concentrations and dilutions using an Amicon Ultra-15 centrifugal filter device (Mr 30,000 cutoff; Millipore Corp.). Step 4: Q-Sepharose—The enzyme solution was applied to a Q-Sepharose HP column (1.6 × 31.5 cm; GE Healthcare) equilibrated with buffer S1 at a flow rate of 1.5 ml/min using an AöKTApurifier apparatus. The column was washed with the same buffer. The enzyme was eluted with a linear gradient of 0-0.42 m NaCl in 600 min in buffer S1. The active fractions were combined, concentrated, and equilibrated with buffer S1 by ultrafiltration as described above. Step 5: Phenyl-Sepharose—Ammonium sulfate was added to the enzyme solution to 20% saturation. The enzyme solution was applied to a phenyl-Sepharose HP column (1.0 × 29 cm; GE Healthcare) equilibrated with 20 mm Tris-HCl (pH 8.5) containing 20% saturated ammonium sulfate at a flow rate of 1 ml/min using anAöKTApurifier apparatus. The column was washed with the same buffer. The enzyme was eluted with a linear gradient (0-100% in 480 min) of 20 mm Tris-HCl (pH 8.5) containing 50% (v/v) ethylene glycol. The active fractions were combined, concentrated, and equilibrated with buffer A (20 mm potassium phosphate (pH 8.0) containing 0.1% (w/v) CHAPS and 50 μm UDP-glucose) as described above. Step 6: Hydroxylapatite Chromatography—The enzyme solution was applied to a hydroxylapatite column (1.0 × 12.5 cm; Bio-Rad) equilibrated with buffer A at a flow rate of 1 ml/min using anAöKTApurifier apparatus. The column was washed with the same buffer. The enzyme was eluted with a linear gradient (0-25% in 200 min) of 0.4 m potassium phosphate (pH 8.0) containing 0.1% (w/v) CHAPS and 50 μm UDP-glucose. The active fractions were combined, concentrated, and equilibrated with buffer B (20 mm Tris-HCl (pH 8.5) containing 0.15 m NaCl, 0.1% (w/v) CHAPS, and 50 μm UDP-glucose) as described above. Step 7: Gel Filtration—The enzyme solution was applied to a HiLoad 26/60 Superdex 200 prep grade column (GE Health-care) equilibrated with buffer B and eluted at a flow rate of 1 ml/min using anAöKTApurifier apparatus. The active fractions were combined, concentrated, and re-chromatographed under the same conditions. The active fractions were combined, concentrated, and equilibrated with buffer S2 (20 mm Tris-HCl (pH 8.5) containing 0.1% (w/v) CHAPS and 50 μm UDP-glucose) as described above. Step 8: Resource Q—The enzyme solution was applied to a Resource Q column (6 ml; GE Healthcare) equilibrated with buffer S2 at a flow rate of 1 ml/min using anAöKTApurifier apparatus. The column was washed with the same buffer. The enzyme was eluted with a linear gradient of 0-0.24 m NaCl in 90 min in buffer S2. The active fractions were combined and subjected to re-chromatography on Resource Q under the same conditions. The active fractions were concentrated and equilibrated with buffer C (25 mm bis-Tris/iminodiacetic acid (pH 7.1) containing 0.1% (w/v) CHAPS and 50 μm UDP-glucose). Step 9: Chromatofocusing on Mono P—The enzyme solution was applied to a Mono P column (0.5 × 20 cm; GE Healthcare) equilibrated with buffer C at a flow rate of 0.5 ml/min using an AöKTApurifier apparatus. The column was washed with the same buffer. The enzyme was eluted with 10% (v/v) Polybuffer 74 (GE Healthcare)/iminodiacetic acid (pH 4.0) containing 0.1% (w/v) CHAPS and 50 μm UDP-glucose for 120 min. Fractions (0.75 ml each) were collected in a 96-well DeepWell plate (Greiner Bio-One International AG, Frickenhausen, Germany) containing 0.25 ml of 1 m Tris-HCl (pH 8.5) to avoid prolonged exposure of the enzyme to acidic pH. The active fractions were combined, concentrated, and equilibrated with buffer S3 (20 mm Tris-HCl (pH 8.5) containing 0.1% (w/v) CHAPS) as described above, thereby eliminating Polybuffer 74 from the purified enzyme. SDS-PAGE was carried out according to the method of Laemmli (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207529) Google Scholar). The proteins on the gels were visualized by silver staining or with Coomassie Brilliant Blue R-250. Degenerate oligonucleotide primers were designed based on the partial amino acid sequences determined (see “Results and Discussion”): IF1, 5′-GCIATHGTIATHGAYTTYATGAA-3′; IF2, 5′-TTYATGAAYTTYAAYGAYCCIAA-3′; IR1, 5′-TCICKIACICKRTCICCIARYTCIGT-3′, and IR2, 5′-TCIGTISWISWIACRAAICCRTC-3′. The total RNA was isolated from the roots of G. max seedlings using an RNeasy plant mini kit (Qiagen Inc., Hilden, Germany). Reverse transcription-PCR was performed using a Qiagen OneStep reverse transcription-PCR kit with primers IF1 and IR1 and total RNA from the roots of G. max seedlings. Thermal cycling conditions were as follows. The reverse transcription-PCR mixture was incubated at 50 °C for 30 min for the reverse transcriptase reaction, followed by 35 cycles of PCR (one cycle consisted of 94 °C for 30 s, 42 °C for 30 s, and 72 °C for 1 min) and incubation at 72 °C for 10 min. An amplified fragment was further subjected to nested PCR using primers IF2 and IR2. Thermal cycling conditions were 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 1 min and, finally, 72 °C for 10 min. The amplified fragment, which was ∼1 kilobase pair in length, was cloned into pCR2.1-TOPO (Invitrogen) and subjected to sequencing using a dye terminator cycle sequencing kit (Beckman Coulter, Fullerton, CA) with a CEQ 2000 DNA analysis system (Beckman Coulter). The 5′-fragment was obtained using a 5′-RACE system (Invitrogen) with primers 5GSP1 (5′-GGATTTGCAAAGGTTGGTCTGTATCC-3′), 5GSP2 (5′-GAGGGTTGGGTGAATGGTGGG-3′), and 5GSP3 (5′-GTGAATGGTGGGGTAGTAAAGGAG-3′) and total RNA from the roots of G. max seedlings. The 3′-fragment was obtained using SuperScript™ II reverse transcriptase (Invitrogen) and KOD-Plus (Toyobo Co., Ltd., Osaka) with primers 3GSP1 (5′-GGGTTGTGGGAACCGAGTTGG-3′), 3GSP2 (5′-GTGCTGATGACTCGGCGGAG-3′), oligo(dT) primer, and total RNA from the roots of G. max seedlings. The internal fragment was obtained using SuperScript™ II reverse transcriptase and KOD-Plus with primers F400 (5′-GCCCTCACTGAAAATCTCAACAAC-3′) and R1160 (5′-CTTCACCATAACCATCCTGTTCAT-3′) and total RNA from the roots of G. max seedlings. To obtain the full-length GmIF7GT cDNA, recombinant PCR amplification was performed using the 5′-fragment, 3′-fragment, and internal fragment as templates with primers 5Nd (5′-CATATGAAAGACACCATTGTTCTATACCC-3′, with the NdeI site underlined) and 3Ba (5′-AACGGATCCTCAACTCTGTTTCCACAGCTTAG-3′, with the BamHI site underlined). The amplified fragment was cloned into the pCR4Blunt-TOPO vector using a kit (Zero Blunt TOPO PCR cloning kit for sequencing, Invitrogen) and sequenced to confirm the absence of PCR errors. The plasmid was digested with NdeI and BamHI, and the resulting DNA fragment was ligated with the pET-15b vector (Novagen) that had been previously digested with NdeI and BamHI to obtain plasmid pET-15b-GmIF7GT, which encodes an N-terminal in-frame fusion of GmIF7GT with a His6 tag. The resultant plasmid was transformed into Escherichia coli BL21(DE3) cells. The transformant cells were precultured at 37 °C for 16 h in LB broth containing 50 μg/ml ampicillin. Ten milliliters of the preculture was then inoculated into 2000 ml of the same medium. After cultivation at 20 °C until the absorbance at 600 nm reached 0.5, isopropyl β-d-thiogalactopyrano-side was added to the broth at a final concentration of 0.4 mm, followed by further cultivation at 20 °C for 15 h. All subsequent operations were conducted at 0-4 °C. The recombinant E. coli cells were harvested by centrifugation at 5000 × g for 15 min, washed with distilled water, and resuspended in buffer D (20 mm sodium phosphate (pH 7.4) containing 56 mm 2-mercaptoethanol and 0.5 m NaCl). The cells were disrupted at 4 °C by 10 cycles of ultrasonication (where one cycle corresponded to 10 kHz for 1 min, followed by an interval of 1 min) or using a Multi-Beads Shocker Model MBS200 apparatus (Yasui Kikai Corp, Osaka). The cell debris was removed by centrifugation at 5000 × g for 15 min. Polyethyleneimine was slowly added to the supernatant solution to a final concentration of 0.12% (v/v). The mixture was allowed to stand at 4 °C for 30 min, followed by centrifugation at 5000 × g for 15 min. The supernatant was applied to a HisTrap™ HP column (1 ml; GE Healthcare) equilibrated with buffer D. The column was washed with buffer D, and the enzyme was eluted with buffer D containing 0.2 m imidazole. The active fractions were collected, concentrated, and equilibrated with buffer S1 by ultrafiltration as described above. The resulting enzyme solution was applied to a Resource Q column (6 ml) equilibrated with buffer S1 at a flow rate of 1 ml/min using anAöKTApurifier apparatus. The column was washed with the same buffer. The enzyme was eluted with a linear gradient of 0-0.3 m NaCl in buffer S1. The enzyme solution was concentrated and extensively dialyzed at 4 °C against buffer S3 to remove imidazole. In vitro mutagenesis of the GmIF7GT gene was carried out by recombinant PCR with plasmid pET-15b-GmIF7GT (see above) as a template and a specific mutagenesis oligonucleotide primer (data not shown) and primer F400 (see above for nucleotide sequence) or the T7 terminator primer (5′-TGCTAGTTATTGCTCAGCGG-3′) to obtain the site-directed mutants H15A, D125A, H359A, H368A, E376A, E392A, E456A, and E392D. The amplified fragments were digested with XhoI and BamHI, and the resulting DNA fragments were ligated with pET-15b-GmIF7GT that had been previously digested with XhoI and BamHI. Individual mutation was verified by DNA sequencing on both strands. The NΔ49 mutant was prepared by a PCR-based strategy. The GmIF7GT mutants were expressed in E. coli BL21(DE3) transformant cells and purified essentially as described above. The IFGT activity in the crude extract of roots from G. max seedlings was extremely unstable to oxidation, and the addition of 2-mercaptoethanol was essential for its efficient purification. Two activity peaks of IFGT were identified by DEAE-Sepharose column chromatography (Step 3) (Table 1). These activity peaks were completely separated from each other by hydroxylapatite chromatography (Step 6), where the first eluted peak showed higher specific activity than the second one. In this study, the first eluted IFGT was further purified. The addition of CHAPS to purification buffers was also essential for purification of this IFGT activity after Step 6, otherwise the activity was irreversibly lost during column chromatography, probably because of irreversible adsorption of the enzyme to matrices of purification resins. Finally, the enzyme (termed GmIF7GT) could be purified to homogeneity with an activity yield of 2.6% after nine purification steps (Fig. 2 and Table 1). The apparently high degree of the present purification (14,000-fold) should not arise from possible underestimation of IFGT activities due to the contaminating activity of the isoflavone conjugate-hydrolyzing β-glucosidase (10Suzuki H. Takahashi S. Watanabe R. Fukushima Y. Fujita N. Noguchi A. Yokoyama R. Nishitani K. Nishino T. Nakayama T. J. Biol. Chem. 2006; 281: 30251-30259Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) in the crude extracts because this β-glucosidase was inert at the pH employed for IFGT assay (pH 8.5). Purified GmIF7GT showed a single protein band with a molecular mass of 45 kDa on SDS-polyacrylamide gels (Fig. 2). The native molecular mass of purified GmIF7GT was estimated to be 46 kDa by gel filtration chromatography on a Superdex 200 prep grade column, indicating that the enzyme is monomeric. The N-terminal amino acid sequence of the purified enzyme was determined by automated Edman degradation to be Thr-Thr-Thr-Leu-Ala-Cys-Asp-Ser-Asn-Ala-Gln-Tyr-Ile-Ala (termed sequence 1) (supplemental Fig. 1S). To obtain the internal amino acid sequences of the purified protein, it was digested with a lysyl endopeptidase from Achromobacter lyticus M497-1 (Wako, Tokyo, Japan), and the resulting peptides were separated by reversed-phase HPLC as described previously (27Nakayama T. Yonekura-Sakakibara K. Sato T. Kikuchi S. Fukui Y. Fukuchi-Mizutani M. Ueda T. Nakao M. Tanaka Y. Kusumi T. Nishino T. Science. 2000; 290: 1163-1166Crossref PubMed Scopus (182) Google Scholar). The amino acid sequences of some of these peptides were determined to be Ala-Ile-Val-Ile-Asp-Phe-Met-Asn-Phe-Asn-Asp-Pro-Lys (sequence 2), Val-Ala-Leu-Ala-Val-Asn-Glu-Asn-Lys (sequence 3), Asp-Gly-Phe-Val-Ser-Ser-Thr-Glu-Leu-Gly-Asp-Arg-Val-Arg-Glu (sequence 4), and Leu-Trp-Lys (sequence 5).TABLE 1Purification of GmlF7GT from the roots of G. max seedlingsStepTotal proteinTotal activitySpecific activityPurificationRecoverymgpicokatalspicokatals/mg protein-fold%1. Crude extract490076001.61.01002. Ammonium sulfate fractionation150063004.22.6833. DEAE-Sepharose17049002918644. Q-Sepharose5441007648545. Phenyl-Sepharose19280015094376. Hydroxylapatite8.22500310190337. Superdex 2000.1999052003300138. Resource Q0.01837021,00013,0004.99. Mono P0.00920022,00014,0002.6 Open table in a new tab Reaction of purified GmIF7GT with genistein yielded a single transfer product, which coeluted with genistein 7-O-β-d-glucopyranoside (genistin) in analytical reversed-phase HPLC (data not shown). The 1H NMR spectra of the glucosylated product were identical to those of authentic genistin. These results indicate that the purified enzyme catalyzes the regiospecific transfer of the glucosyl group to a 7-hydroxy group of the