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Originally published In Press as doi:10.1074/jbc.M702651200 on June 11, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23581-23590, August 10, 2007
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A UDP-Glucose:Isoflavone 7-O-Glucosyltransferase from the Roots of Soybean (Glycine max) Seedlings

PURIFICATION, GENE CLONING, PHYLOGENETICS, AND AN IMPLICATION FOR AN ALTERNATIVE STRATEGY OF ENZYME CATALYSIS*Formula

Akio Noguchi{ddagger}, Atsushi Saito{ddagger}, Yu Homma{ddagger}, Masahiro Nakao§, Nobuhiro Sasaki{ddagger}, Tokuzo Nishino{ddagger}, Seiji Takahashi{ddagger}, and Toru Nakayama{ddagger}1

From the {ddagger}Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11, Sendai 980-8579, Japan and the §Suntory Research Center, Mishima-gun, Shimamoto-cho, Osaka 618-8503 Japan

Received for publication, March 28, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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-beta-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 (1, 2). They are also involved in defensive mechanisms of legumes against pathogen infection (3, 4). Moreover, isoflavones exhibit a wide variety of bioactivities that are beneficial to human health (5).

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) (6, 7). 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 (8). The resulting isoflavone aglycons are 7-O-glucosylated and subsequently 6''-O-malonylated (9) to produce isoflavone conjugates, which then accumulate in vacuoles (3). The release of aglycons from these conjugates is catalyzed by an isoflavone conjugate-hydrolyzing beta-glucosidase. We showed previously that, in G. max seedlings, this beta-glucosidase is localized exclusively in the root apoplast (10). Most biological and biomedical activities of isoflavones have been identified with their aglycon forms (4, 5, 11), and conjugation can affect the pharmacokinetics of the dietary isoflavonoids (5). 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)2 (Fig. 1) (3). 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 (12, 13). So far, glycosyltransferases involved in plant secondary metabolism (i.e. plant secondary product glycosyltransferases (PSPGs)) have all been grouped into glycosyltransferase family 1 (14-19). These PSPGs are characterized by a unique well conserved sequence of ~45 amino acid residues (called a PSPG box (14)) and a catalytic mechanism leading to an inversion of the anomeric configuration of transferred sugar (12). Recent crystallographic studies of PSPGs from Medicago truncatula (UGT71G1) (20) and Vitis vinifera (VvGT1) (21), along with the results of site-directed mutagenesis studies of these enzymes (22), 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.


Figure 1
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  		FIGURE 1.
GmIF7GT-catalyzed transfer of a glucosyl group to isoflavones. 1, R=H, daidzein; R=OH, genistein. 2, R=H, daidzin; R=OH, genistin.

 
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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials and Chemicals
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.

Enzyme and Protein Assays
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 x 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 (23) using bovine serum albumin as a standard. The concentration of purified GmIF7GT was determined by absorption coefficients {epsilon}280 = 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.

Enzyme Kinetics
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 (24, 25).

Purification of GmIF7GT from the Roots of G. max Seedlings
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 x 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 x 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 x 51.5 cm; GE Healthcare) equilibrated with buffer S1 at a flow rate of 2 ml/min using anÄKTApurifier 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 x 31.5 cm; GE Healthcare) equilibrated with buffer S1 at a flow rate of 1.5 ml/min using an ÄKTApurifier 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 x 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 anÄKTApurifier 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 x 12.5 cm; Bio-Rad) equilibrated with buffer A at a flow rate of 1 ml/min using anÄKTApurifier 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 anÄKTApurifier 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 anÄKTApurifier 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 x 20 cm; GE Healthcare) equilibrated with buffer C at a flow rate of 0.5 ml/min using an ÄKTApurifier 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 (26). The proteins on the gels were visualized by silver staining or with Coomassie Brilliant Blue R-250.

cDNA Cloning and Heterologous Expression of GmIF7GT
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 SuperScriptTM 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 SuperScriptTM 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 beta-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 x 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 x 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 x g for 15 min. The supernatant was applied to a HisTrapTM 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 anÄKTApurifier 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.

Site-directed Mutagenesis and Expression
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 N{Delta}49 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.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Purification of GmIF7GT from the Roots of G. max Seedlings
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 beta-glucosidase (10) in the crude extracts because this beta-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 (27). 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).


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  		TABLE 1
Purification of GmlF7GT from the roots of G. max seedlings

 


Figure 2
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  		FIGURE 2.
SDS-PAGE of GmIF7GT preparations obtained during purification of the enzyme from the roots of G. max seedlings. The lane numbers correspond to purification steps described in Table 1, with lanes 7-1 and 7-2 indicating the enzyme preparations after the first and second Superdex 200 chromatography steps and lane M indicating the molecular mass markers. The enzyme preparations and marker proteins were simultaneously electro-phoresed under the conditions described by Laemmli (26) and silver-stained.

 
Catalytic Properties and Stability
Reaction of purified GmIF7GT with genistein yielded a single transfer product, which coeluted with genistein 7-O-beta-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 substrate isoflavone (Fig. 1). The calculated kcat value for GmIF7GT-catalyzed glucosyl transfer to genistein was 0.74 ± 0.02 s-1, and the Km values for genistein and UDP-glucose were 3.6 ± 0.4 and 190 ± 50 µM, respectively (Table 2). The purified enzyme was active over a pH range of 7.0-9.0 with maximum activity at 8.5 (at 30 °C) and was stable at pH 5.5-10.0 (at 20 °C for 8 h) and below 30 °C (at pH 8.5 for 1 h) (supplemental Fig. 2S).


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  		TABLE 2
Kinetic parameters for the enzyme from the roots of G. max seedlings, recombinant wild-type enzyme, and mutants

Values in parentheses indicate the relative percent of the kcat and kcat/Km values of mutants, with that of the recombinant wild-type enzyme taken as 100%.

 
Substrate Specificity
Glycosyl Donor Specificity—The glycosyl donor specificity of purified GmIF7GT was examined with UDP-glucose, UDP-glucuronic acid, and UDP-galactose using genistein as a glycosyl acceptor. The relative activities for UDP-glucuronic acid and UDP-galactose were 0 and 12% of the activity for UDP-glucose, respectively, indicating that the enzyme is specific for UDP-glucose.

Glucosyl Acceptor Specificity—The glucosyl acceptor specificity of purified GmIF7GT was examined with a wide variety of phenolics, including flavonoids, coumarins, phenylpropanoids, and benzoic acid derivatives, using UDP-glucose as the glucosyl donor. The enzyme showed the highest activity with genistein. It also showed appreciable activities toward the following flavonoids and coumarins: daidzein (relative activity for genistein, 73%), formononetin (67%), quercetin (32%), kaempferol (19%), 4,2',4',6'-tetrahydoxychalcone (15%), apigenin (14%), aureusidin (5%), esculetin (4%), and naringenin (3%) (supplemental Fig. 3S). HPLC analysis showed that the reaction of the purified enzyme with daidzein, aureusidin, or naringenin gave rise to a single transfer product, which coeluted with the corresponding 7-O-glucoside (or 6-O-glucoside for aureusidin) (data not shown). None of the following phenolics served as glucosyl acceptors: cyanidin, trans-p-coumaric acid, caffeic acid, benzoic acid, m- and p-hydroxybenzoic acids, salicylic acid, salicyl alcohol, and hydroquinone.

Effects of Metal Ions and Enzyme Inhibitors
The effects of divalent metal ions (as chlorides), a metal chelator (EDTA), compounds that are structurally related to substrates and products, and enzyme inhibitors on GmIF7GT activity were examined. The enzyme activity was partially inhibited by 0.1 mM Fe2+ (residual activity, 28%) and 0.1 mM Hg2+ (26%) ions. Other 0.1 mM metal ions (Ca2+, Cd2+, Co2+, Cu2+, Mg2+, Mn2+, Ni2+, Sn2+, and Zn2+; final concentration), 1 mM EDTA, 1 mM uridine, 1 mM UMP, 1 mM UDP, 1 mM UTP, 1 mM glucose, 1 mM diethyl pyrocarbonate (histidine-modifying agent), and 1 mM phenylmethanesulfonyl fluoride (serine-modifying agent) had negligible effects on the catalytic activity (residual activity in excess of 64%).

cDNA Cloning, Heterologous Expression, and Sequence Comparison
Degenerate PCR primers (IF1, IF2, IR1, and IR2; see "Experimental Procedures" for nucleotide sequences) were designed based on the partial amino acid sequences determined (supplemental Fig. 1S). Primers IF1 and IR1 were used in reverse transcription-PCR with total RNA extracted from the roots of G. max seedlings as a template. The amplified DNA fragment was further subjected to nested PCR using primers IF2 and IR2, yielding a PCR product of 890 bp encoding partial amino acid sequences that had been determined from the purified enzyme. The full-length GmIF7GT cDNA was obtained by recombinant PCR with the 5'- and 3'-end fragments that had been obtained by 5'- and 3'-RACE, respectively.

The full-length GmIF7GT cDNA contained an open reading frame of 1422 bp encoding a protein of 474 amino acid residues (calculated molecular mass, 52,035 Da). A comparison of the deduced amino acid sequence and N-terminal amino acid sequence of the purified enzyme (see above) showed that the N terminus of the purified enzyme starts with Thr-50; therefore, the purified enzyme was devoid of an N-terminal 49-residue portion of the cDNA-encoded amino acid sequence. The internal amino acid sequences determined for the purified enzyme were also identified (supplemental Fig. 1S).

Full-length GmIF7GT (474 amino acids in length) was expressed under the control of the T7 lac promoter in E. coli BL21(DE3) cells as a soluble, catalytically active protein at 2.4 mg/liter of culture broth. Recombinant GmIF7GT could be easily purified to homogeneity from crude extracts of the transformant cells with an activity yield of 38%. The glucosyl donor and acceptor specificities of recombinant GmIF7GT were indistinguishable from those of the enzyme purified from G. max seedling roots (the truncated form) (Table 2 and supplemental Fig. 3S), allowing us to further confirm that the cloned cDNA codes for GmIF7GT. The other enzymologic properties of the recombinant enzyme (supplemental Fig. 2S) were also consistent with the fact that this cDNA codes for GmIF7GT.

The deduced amino acid sequence of GmIF7GT (supplemental Fig. 1S) contains several conserved amino acids of the PSPG box (see above) and shows a significant similarity to those of PSPGs in glycosyltransferase family 1. According to the glycosyltransferase nomenclature guidelines (28, 29), the systematic name of GmIF7GT is UGT88E3. Among biochemically characterized PSPGs, the highest sequence similarity was found with UDP-glucose:chalcone 4'-O-glucosyltransferases from Antirrhinum majus (AmC4'GT (UGT88D3); DDBJ/Gen-BankTM/EBI accession number AB198665; 42% identity) (30) and Linaria vulgaris (LvC4'GT (UGT88D2); accession number BAE48240 [GenBank] ; 41% identity) (31), Scutellaria baicalensis UDP-glucuronate:baicalein 7-O-glucuronosyltransferase (SbB7GAT; accession number BAC98300 [GenBank] ; 41% identity), and Dorotheanthus bellidiformis betanidin 6-O-glucosyltransferase (accession number AAL57240 [GenBank] ; 31% identity) (32). It also shows high sequence similarities (60-71% identity) to PSPG homologs of unknown biochemical function (Vigna angularis (adzuki beans) glucosyltransferase-1, -3, and 5; accession numbers BAB86919 [GenBank] , BAB86921 [GenBank] , and BAB86923 [GenBank] , respectively). Notably, however, GmIF7GT shows only low sequence similarity (29% identity) to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase (GeIF7GT (UGT73F1); accession number BAC78438 [GenBank] ) (33), which displays a substrate specificity that is very similar to that of GmIF7GT. GmIF7GT is 32 and 25% identical in primary structure to PSPGs from M. truncatula (UGT71G1; accession number AAW56092 [GenBank] ) and V. vinifera (VvGT1; accession number AAB81682 [GenBank] ), respectively, the crystal structures of which were clarified recently (20, 21).

A phylogenetic tree of functionally characterized PSPGs shows several clusters (Fig. 3) (14, 18, 34), which appear to be characterized by the specificity of the flavonoid glycosyltransferase activities of enzymes involved therein. For example, Clusters I, II, and III (Fig. 3) are characterized by flavonoid 3-O-glycosyltransferases (UGT78 PSPGs), flavonoid 5-O-glycosyltransferases (UGT75 PSPGs), and flavonoid 7-O-glycosyltransferases (UGT73 PSPGs, including GeIF7GT, and UGT89 PSPGs), respectively (14, 34, 35). Cluster IV contains PSPGs catalyzing the glycosyl transfer to the sugar moiety of flavonoid glycosides (19). Notably, despite the fact that GmIF7GT is essentially a flavonoid 7-O-glycosyltransferase, it is distantly related to Cluster III and appears to form a new cluster together with AmC4'GT, LvC4'GT, SbB7GAT, Rosa hybrida UDP-glucose:anthocyanidin 5,3-O-glucosyltransferase, and other functionally unknown PSPGs (Fig. 3). Many of these PSPGs have also been assigned as UGT88 enzymes. AmC4'GT, LvC4'GT, and SbB7GAT are flavonoid-specific PSPGs that catalyze the glycosyl transfer to the position of flavonoids equivalent to the 7-position of isoflavones (i.e. the 4'-position of chalcones (for AmC4'GT and LvC4'GT) and to the 7-position of flavones (for SbB7GAT)) (30, 31, 36), which is detailed in supplemental Fig. 4S. R. hybrida UDP-glucose:anthocyanidin 5,3-O-glucosyltransferase appears to be exceptional among these PSPGs because it catalyzes two successive glucosylations at the 5- and 3-positions of anthocyanidins (37). Thus, the UGT88-related PSPGs include enzymes showing a regiospecificity of glycosyl transfer for the 7-position of isoflavones and the corresponding position of other flavonoids. These enzymes are phylogenetically distant from the Cluster III flavonoid 7-O-glycosyltransferases such as GeIF7GT and UGT89C1.

It should be mentioned that GmIF7GT is most likely a cytoplasmic enzyme, judging from the absence of a predictable signal sequence and membrane-spanning domain in the full-length GmIF7GT sequence as well as the biochemical properties of GmIF7GT such as optimum pH for activity and pH stability (see above). However, it is possible that this enzyme is associated with the endomembrane system as a peripheral component, as suggested previously (38). The significance of the loss of the N-terminal 49-residue segment in purified GmIF7GT remains to be clarified. To our knowledge, such truncation of an N-terminal peptide has not been reported for other PSPGs. Note that, during the purification of GmIF7GT from crude extracts of the roots of G. max seedlings, a minor peak of IFGT activity was separated from GmIF7GT (the truncated form) in hydroxylapatite chromatography (see above). This activity peak might arise from the full-length form of GmIF7GT because the estimated molecular mass of the peak (~50 kDa) was slightly higher than that of the purified enzyme as analyzed by Superdex 200 gel filtration chromatography. The pH stability and thermostability of the purified enzyme (the truncated form) were noticeably higher than those of recombinant full-length GmIF7GT (supplemental Fig. 2S), implying that this truncation might be of physiological significance in producing a stable mature form of the enzyme.


Figure 3
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  		FIGURE 3.
Non-rooted phylogenetic tree of PSPGs. The sequences used for the alignment are primarily those of PSPGs that are characterized functionally, but sequences of PSPGs that are not yet functionally characterized, although closely related to GmIF7GT, are also included (i.e. UGT88A1; Oryza sativa putative glucosyltransferase; Trifolium pratense putative glucosyltransferase; V. angularis glucosyltransferase-1, -3, and -5; and Stevia rebaudiana UDP-glycosyltransferase 88B1). Registered UGT88 names according to the glycosyltransferase nomenclature guidelines (28, 29) are shown in parentheses. The tree was constructed from a ClustalW program multiple alignment using a neighbor-joining method of TreeView (42). Bar = 0.1 amino acid substitution/site. Numbers indicate bootstrap values >800. Known clusters (I, II, III, and IV) of PSPGs (14, 19, 34) are shown with open circles, whereas the UGT88-related PSPGs are enclosed in a gray circle. The names and DDBJ/GenBankTM/EBI accession numbers of PSPGs used for the alignment are as follows: AtF3G7GT, A. thaliana UDP-glucose: flavonol-3-O-glycoside 7-O-glucosyltransferase (UGT73C6; accession number Q9ZQ95); AtF7GT, A. thaliana flavonoid 7-O-glucosyltransferase (UGT73B1; accession number AAL90934); AtF5GT, A. thaliana flavonoid 5-O-glucosyltransferase (UGT75C1; accession number AAM91686); AtF3GT, A. thaliana flavonoid 3-O-glucosyltransferase (UGT78D2; accession number AAM91139); AmC4'GT, A. majus UDP-glucose:chalcone 4'-O-glucosyltransferase (UGT88D3; accession number AB198665); BpUGAT, Bellis perennis UDP-glucuronic acid:anthocyanidin-3-O-glucoside 2''-O-glucuronosyltransferase (UGT94B1; accession number AB190262); CmF7G12RT, Citrus maxima UDP-rhamnose:flavonoid-7-O-glycoside 1,2-O-rhamnosyltransferase (accession number AAL06646); DbB5GT, D. bellidiformis betanidin 5-O-glucosyltransferase (UGT73A5; accession number CAB56231); DbB6GT, D. bellidiformis betanidin 6-O-glucosyltransferase (accession number AB198665); FiF3GT, Forsythia x intermedia UDP-glucose:flavonoid 3-O-glucosyltransferase (accession number AAD21086); GeIF7GT, G. echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase (accession number BAC78438); GmIF7GT, G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (UGT88E3; this study; accession number AB292164); GtF3GT, Gentiana triflora flavonoid 3-O-glucosyltransferase (UGT78B1; accession number BAA12737); HvF3GT, Hordeum vulgare UDP-glucose:flavonoid 3-O-glucosyltransferase (accession number CAA33729); IpA3G2GT, Ipomoea purpurea UDP-glucose:anthocyanidin-3-glucoside 2''-O-glucosyltransferase (UGT79G16; accession number BAD95882); Letwi1, Lycopersicon esculentum probable glucosyltransferase twi1 (accession number T07404); LvC4'GT, L. vulgaris UDP-glucose:chalcone 4'-O-glucosyltransferase (accession number BAE48240); NtGT2, N. tabacum glucosyltransferase-2 (accession number BAB88935); NtGT3, N. tabacum glucosyltransferase-3 (accession number BAB88934); NtJAGT, N. tabacum jasmonate-induced glucosyltransferase (accession number T02238); NtSAGT, N. tabacum UDP-glucose:salicylic acid glucosyltransferase (accession number AAF61647); OsGT, Oryza sativa putative glucosyltransferase (accession number BAC10743); PfF3GT, Perilla frutescens UDP-glucose:flavonoid 3-O-glucosyltransferase (accession number BAA19659); PfA5GT, P. frutescens UDP-glucose:anthocyanin 5-O-glucosyltransferase (accession number BAA36421); PhF3GT, P. hybrida UDP-glucose:anthocyanin 3-O-glucosyltransferase (accession number BAA89008); PhA5GT, P. hybrida UDP-glucose:anthocyanin 5-O-glucosyltransferase (accession number BAA89009); PhA3GRT, P. hybrida UDP-rhamnose:anthocyanidin-3-O-glycoside rhamnosyltransferase (accession number CAA50376); RhA53GT, R. hybrida UDP-glucose: anthocyanidin 5,3-O-glucosyltransferase (accession number BAD99560); RsAS, Rauvolfia serpentina hydroquinone glucosyltransferase (arbutin synthase; accession number CAC35167); SbB7GAT, S. baicalensis UDP-glucuronate:baicalein 7-O-glucuronosyltransferase (accession number BAC98300); SbF7GT, S. baicalensis UDP-glucose:flavonoid 7-O-glucosyltransferase (accession number BAA83484); SmGT, Solanum melongena glycosyltransferase (accession number CAA54558); SrGT88B1, S. rebaudiana UDP-glycosyltransferase 88B1 (UGT88B1; accession number AAR06919); ThA5GT, Torenia hybrida UDP-glucose:anthocyanin 5-O-glucosyltransferase (accession number BAC54093); TOGT1, N. tabacum phenylpropanoid:glucosyltransferase-1 (accession number AAK28303); TOGT2, N. tabacum phenylpropanoid:glucosyltransferase-2 (accession number AAK28304); TpGT, Trifolium pratense putative glucosyltransferase (accession number BAE71309); UGT71G1, M. truncatula triterpene UDP-glucosyltransferase (accession number AAW56092); UGT88A1, A. thaliana putative glucosyltransferase (accession number AAN28841); UGT89C1, A. thaliana flavonol 7-O-rhamnosyltransferase (accession number AAP31923); VaABAGT, V. angularis abscisic acid glucosyltransferase (accession number BAB83692); VaGT1, V. angularis glucosyltransferase-1 (accession number BAB86919); VaGT3, V. angularis glucosyltransferase-3 (accession number BAB86921); VaGT5, V. angularis glucosyltransferase-5 (accession number BAB86923); VhA5GT, Verbena x hybrida UDP-glucose:anthocyanin 5-O-glucosyltransferase (accession number BAA36423); VvGT1, V. vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase-1 (accession number AAB81682).

 


Figure 4
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  		FIGURE 4.
Comparison of the amino acid sequences containing the conserved amino acid residues in PSPGs. The DDBJ/GenBankTM/EBI accession numbers of the enzymes are provided in the legend to Fig. 3. The mutated sites are indicated by asterisks. VaGT5, V. angularis glucosyltransferase-5; RhA53GT, R. hybrida UDP-glucose:anthocyanidin 5,3-O-glucosyltransferase; PhA5GT, P. hybrida UDP-glucose:anthocyanin 5-O-glucosyltransferase; BpUGAT, B. perennis UDP-glucuronic acid:anthocyanidin-3-O-glucoside 2''-O-glucuronosyltransferase; RsAS, R. serpentina hydroquinone glucosyltransferase (arbutin synthase).

 
Exploring Amino Acid Residues That Are Important for GmIF7GT Catalysis
Recent structural elucidation of PSPGs and mutagenesis studies permitted the identification of amino acid residues that are functionally important for PSPG catalysis (20, 21), which include His-15* and Asp-125* (Fig. 4). (Amino acid residues are numbered so as to correspond to the numbering of GmIF7GT on the basis of the alignment shown in Fig. 4, and positional numbering, according to this notation, is indicated by an asterisk.) In the crystal structures of VvGT1 complexed with kaempferol (a glycosyl acceptor) and UDP-2-deoxy-2-fluoro-glucose (a glycosyl donor analog), His-15* is hydrogen-bonded to the 3-hydroxy group of kaempferol, which serves as a nucleophile that attacks the anomeric center of the glycosyl donor molecule (21). Consistent with this observation, replacement of His-15* with alanine results in a complete loss of catalytic activity (21). These observations led to the proposal that, in VvGT1 catalysis, His-15* plays the role of a general base for deprotonation of hydroxy function of acceptor molecules to facilitate glycosidic linkage formation (21). Moreover, Asp-125* of VvGT1 is hydrogen-bonded to His-15* and appears to assist the general acid/base role of His-15* during catalysis in a manner reminiscent of a catalytic triad described for serine hydrolases (21). Similar roles of His-15* and Asp-125* during enzyme catalysis have also been proposed for UGT71G1 (20). However, the GmIF7GT preparation obtained from the roots of G. max seedlings, which displayed a high catalytic activity (kcat = 0.7 s-1 at pH 8.5 and 30 °C), was found to be devoid of an N-terminal 49-residue segment (see above) and thus lacked His-15, indicating that His-15 is not important for GmIF7GT catalysis, unlike the case of UGT71G1 and VvGT1. To confirm this, a H15A mutant of full-length GmIF7GT was prepared. Also, an N{Delta}49 mutant of GmIF7GT was prepared, in which the N-terminal 49-residue segment was deleted, mimicking GmIF7GT that was purified from the roots of G. max seedlings. Both of these mutants were expressed in E. coli cells. The H15A mutant was purified to homogeneity by successive nickel affinity and anion exchange chromatography and analyzed for its kinetic properties. The results of kinetic analyses showed that the H15A mutant displayed a considerable kcat value (i.e. 17% of the wild-type value) (Table 2).3 The affinity of the enzyme for UDP-glucose was rather noticeably enhanced upon the H15A substitution, so the catalytic efficiency of H15A for UDP-glucose was comparable with that of the wild-type enzyme (83%). We were unable to purify the N{Delta}49 mutant; most of the expressed product was obtained as inactive inclusion bodies, probably because deletion of the N-terminal portion hampered the correct folding of the enzyme molecule into its catalytically active form. However, the crude extracts of the transformant cells expressing the N{Delta}49 mutant contained a very small amount of a soluble form of the mutant enzyme and displayed a detectable level of IFGT activity. The D125A mutant was also prepared to evaluate the functional significance of Asp-125 for GmIF7GT catalysis. The results showed that D125A displayed a kcat value comparable with that of the wild-type enzyme (87%) (Table 2). Again, the affinity of D125A for UDP-glucose was much higher; hence, the catalytic efficiency of the mutant for UDP-glucose was higher than that of the wild-type enzyme. Therefore, the involvement of Asp-125 during GmIF7GT catalysis is also highly unlikely. All of these results led to the conclusion that GmIF7GT does not utilize His-15 and Asp-125 for its enzyme catalysis. It is noteworthy that His-15* and Asp-125* are replaced by other amino acid residues in some glycosyltransferases; for example, His-15* is replaced by cysteine in Arabidopsis thaliana UGT76C1 (DDBJ/GenBankTM/EBI accession number BAB10792 [GenBank] ) and UGT76C2 (accession number AAN28835 [GenBank] ) (see Ref. 20), and Asp-125* is replaced by arginine in Crocus sativus UGTCs2 (accession number AAP94878 [GenBank] ), serine in Nicotiana tabacum glucosyltransferase-2 (accession number BAB88935 [GenBank] ) (39) and Petunia x hybrida UDP-glucose:anthocyanin 5-O-glucosyltransferase (accession number BAA89009 [GenBank] ) (Fig. 4), and asparagine in Brassica napus UDP-glucose:sinapate glucosyltransferase (accession number AAF98390 [GenBank] ) (40).

To explore amino acid residues that are important for GmIF7GT catalysis, alanine-scanning mutagenesis studies were carried out concerning amino acid residues that are well conserved around PSPG box sequences: His-359, His-368, Glu-376, Glu-392, and Glu-456 (Fig. 4). Among the selected sites of mutagenesis, His-368* was shown to be very important for the catalytic activity of arbutin synthase (41). However, the relative kcat value for H368A was 260% of the wild-type value. In addition, the relative kcat value for H359A was 78% of the wild-type value. Thus, none of the conserved His residues examined in this study (i.e. His-15, His-359, and His-368) appear to play a crucial role in GmIF7GT catalysis. This observation was corroborated by the fact that GmIF7GT activity was insensitive to 1 mM diethyl pyrocarbonate, a histidine modifier (see above). Replacement of either Glu-376 or Glu-456 with alanine caused no significant diminution of the kcat value. Very interestingly, however, replacement of Glu-376 with alanine specifically resulted in a large increase in the Km value for UDP-glucose, consistent with the fact that Glu-376* interacts with the nucleotide moiety of bound UDP-glucose in the crystal structure of a Michaelis complex of UGT71G1 (20). Most importantly, replacement of Glu-392 with alanine resulted in a dramatic decrease in the kcat value, with Km values essentially unchanged (Table 2). Because position 392* of PSPGs is invariably occupied by an acidic amino acid residue (i.e. glutamic or aspartic acid) (Fig. 4), we also prepared E392D to examine its kinetic properties. The mutant unexpectedly displayed a very high kcat value. The Km values of the mutant for genistein and UDP-glucose were both decreased, so the catalytic efficiencies of E392D for both substrates were significantly higher (14- and 22-fold) than those of the wild-type enzyme (Table 2). These results corroborate the importance of a carboxylate group at position 392 for GmIF7GT catalysis. It must be mentioned that, in the crystal structures of VvGT1 and UGT71G1 complexed with UDP-glucose, the amino acid residue at position 392* (aspartic acid in VvGT1 and glutamic acid in UGT71G1) was shown to be hydrogen-bonded with O-3 and O-4 of the glucosyl moiety of the bound UDP-glucose (20, 21). Replacement of this residue with alanine also resulted in a complete loss of catalytic activities of VvGT1 and UGT71G1 (20, 21). In UGT71G1, Glu-392* was proposed to be a key residue for sugar recognition and specificity, although it has recently been proposed that the residues involved in sugar donor specificity of glycosyltransferases cannot be ascribed to a single amino acid residue (34). The observed loss of GmIF7GT activity upon the E392A substitution might be explained in terms of the inability of the mutant to utilize UDP-glucose, given the possible role of Glu-392 in sugar recognition during GmIF7GT catalysis. However, GmIF7GT does not utilize His-15 and Asp-125 during its catalysis (see above) and is therefore expected to utilize a strategy of catalysis that is distinct from that utilized in the catalysis of UGT71G1 and VvGT1; hence, the role of Glu-392 in PSPG catalysis might also be different from that of Glu-392* and Asp-392* in UGT71G1 and VvGT1, respectively. Unlike the case of E376A substitution, replacement of Glu-392 with alanine did not essentially affect the apparent affinity of the enzyme for both substrates, but exclusively affected the kcat value to abolish the catalytic competence of the enzyme. Thus, Glu-392 appears to be involved in primary catalysis, such as general acid/base catalysis, rather than in sugar recognition, during GmIF7GT catalysis. X-ray crystallographic studies of GmIF7GT complexed with substrates and their analogs would be needed to address these issues.

The results obtained in this study might suggest plasticity in the strategy of enzyme catalysis in glycosyltransferases. It would be interesting to examine which PSPGs utilize a strategy of enzyme catalysis similar to that of GmIF7GT catalysis. In this respect, a measure of caution is called for when extrapolating catalytic residues from one glycosyltransferase to another one by means of homology modeling based on scaffolds displaying only remote similarity.

In conclusion, GmIF7GT is a novel member of the UGT88 PSPG group, which includes enzymes showing a regiospecificity of glycosyl transfer for the 7-position of isoflavones and for the corresponding position of other flavonoids. The results obtained in this mutational study indicate that GmIF7GT does not utilize His-15 and Asp-125 during its catalysis and is therefore expected to utilize a strategy that is distinct from that utilized by the catalysis of UGT71G1 and VvGT1. Glu-392 should play a very important role in GmIF7GT catalysis, the details of which should be clarified in future studies. Finally, the GmIF7GT gene obtained in this study will serve as an important tool to engineer isoflavone metabolism because glucosylation plays very important roles in solubilization, accumulation in vacuoles, and mobilization of isoflavonoids in legume cells.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB292164.

* This work was supported in part by a grant from the New Energy and Industrial Technology Development Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains Figs. 1S-4S. Back

1 To whom correspondence should be addressed. Tel. and Fax: 81-22-795-7270; E-mail: nakayama{at}seika.che.tohoku.ac.jp.

2 The 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. Back

3 The H15A mutant of GmIF7GT was purified by nickel affinity chromatography, in which imidazole (0.2 M) was used for specific elution of the mutant from the affinity column. This provides the possibility that the observed catalytic activity of this mutant arose from "chemical rescue," where the added imidazole could bind to the active site of the enzyme to make a surrogate of the imidazole group of His-15. However, this is highly unlikely because the crude extract of E. coli cells expressing the mutant, without added imidazole, displayed strong GmIF7GT activity. The fact that the crude extract of cells expressing the N{Delta}49 mutant showed IFGT activity also corroborates the suggested unimportance of His-15. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Shin-ichi Ayabe (Nihon University) for critical reading of the manuscript. We are grateful to Dr. Eiichiro Ono (Suntory Ltd.) for fruitful discussions concerning the classification and possible biochemical roles of the UGT88 PSPGs. We are also grateful to Prof. Peter Ian Mackenzie (Flinders University School of Medicine) for help with the systematic nomenclature of GmIF7GT and other PSPGs.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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