Molecular cloning, expression and characterization of a ...

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Jae Hyung Ko · Bong Gyu Kim · Hor-Gil Hur ·. Yoongho Lim · Joong-Hoon Ahn. Molecular cloning, expression and characterization of a glycosyltransferase from ...
Plant Cell Rep (2006) 25: 741–746 DOI 10.1007/s00299-006-0119-4

PHYSIOLOGY AND BIOCHEMISTRY

Jae Hyung Ko · Bong Gyu Kim · Hor-Gil Hur · Yoongho Lim · Joong-Hoon Ahn

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Received: 1 September 2005 / Revised: 13 January 2006 / Accepted: 14 January 2006 / Published online: 14 February 2006 C Springer-Verlag 2006 

Abstract Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4 flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized. Keywords Flavonoids . Glycoslytransferase . Oryza sativa

Communicated by I. S. Chung J. H. Ko · B. G. Kim · Y. Lim · J.-H. Ahn () Bio/Molecular Informatics Center, Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, South Korea e-mail: [email protected] Tel.: +82-2-45-3764 Fax: +82-2-3437-6106 e-mail: [email protected] e-mail: [email protected] e-mail: [email protected] H.-G. Hur Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea e-mail: [email protected]

Abbreviations IPTG: Isopropyl-β-d-thiogalactopyranoside . PCR: Polymerase chain reaction . UGT: Uridine diphosphate dependent glycosyltransferase

Introduction Plants produce many types of secondary metabolites, including flavonoids, alkaloids and terpenoids (Wink 1999). These compounds undergo modification reactions such as methylation, hydroxylation and glycosylation, which lead to the structural diversity of secondary metabolites (Schwab 2003). Glycosylation is one of the major modification reactions that often occurs in the final step of the natural compound biosynthesis. The primary roles of glycosylation in plants are: the stabilization of pigments, enhancement of solubility, storage of secondary metabolites and regulation of plant growth regulators. Enzymes leading to glycoside formation known as glycosyltransferases (UGTs), transfer nucleotide-diphosphate-activated sugars to low molecular weight substrates. The activated sugar form is typically UDP-glucose, but UDP-galactose and UDP-rhamnose may also be found. In plants, sugar acceptors include all major classes of secondary metabolites, such as phenolics, terpenoids, cyanohydrins and alkaloids (Vogt and Jones 2000; Jones and Vogt 2001; Bowles et al. 2005). One of the most widely studied classes of plant glycosides is the large and heterogenic group of polyphenols. To date, an overwhelming number of polyphenolic glycosides including flavonoid glycosides have been identified. Flavonoids are an important group of polyphenolic natural products and exhibit a wide range of biological activities including antioxidant and estrogenic properties (Cornwell et al. 2004). Moreover, xenobiotics, defined as foreign compounds and man-made chemicals, may also be glycosylated by the plant (Jones and Vogt 2001). UGTs are so diverse that Arabidopsis has at least 120 UGTs, half of which are theoretically involved in secondary plant metabolism (Li et al. 2001; Ross et al. 2001; Arabidopsis Genome Initiative 2000).

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According to the CAZY database (http://www. afmb. cnrs-mrs.fr/CAZY/), UGTs can be classified into 78 families on the basis of their substrate specificity and sequence similarity (Mackenzie et al. 1997). A total of 193 rice glycosyltransferases belong to Family 1, which is known to be involved in the modification of small compounds such as plant secondary metabolites and hormones. The total number of UGTs in rice is expected to outnumber those found in Arabidopsis. To date, no UGTs from rice have been functionally characterized. This study represents the initial step in the functional analysis of rice UGTs and reports the characterization of RUGT-5, a UGT found in rice.

Materials and methods Cloning of RUGT-5 Total RNA from a rice plant was isolated using Qiagen plant total RNA isolation (Qiagen, Germany, http:// www.qiagen.com). cDNA was synthesized as described by Kim et al. (2003). RUGT-5 was cloned by polymerase chain reaction (PCR) using cDNA as a template. The sequence 5 -gcagcttagctcggagtgac-3 was used as the forward primer, while 5 -gccacctcatacgtcaacag-3 was used as the reverse primer. PCR was performed with Hotstart Taq polymerase (Qiagen, Germany) under the following conditions: 40 cycles of 1 min denaturation at 94◦ C, 1 min annealing at 55◦ C, and 2 min amplification at 72◦ C. The PCR product was subcloned into pGEMT-Easy vector (Promega, USA, http://www.promega.com) and both strands were sequenced. Expression and purification of RUGT-5 in E. coli To express RUGT-5 in E. coli, the full length cDNA of RUGT-5 was amplified with the forward primer containing an EcoRI site and the first 20 nucleotides from the initiation codon of RUGT-5, and with the reverse primer containing the last 20 nucleotides from the stop codon. Pfu DNA polymerase (Intron Biotechnology, Korea, http://www.intron.co.kr) was used for this PCR reaction. The PCR product was digested using EcoRI and sub-cloned into pGEX 5X-1 EcoRI/SmaI sites. The resulting construct was transformed into E. coli BL21(DE3). For the protein induction, the transformant was grown overnight with shaking in 2 ml LB medium containing ampicillin at 37◦ C. The culture was then inoculated in 5 ml fresh LB medium containing ampicillin. It was grown at 37◦ C until absorbance at 600 nm reached to 0.8. IPTG (0.1 mM) was then added. The culture was incubated at 20◦ C with shaking for 20 h. The resulting cell was harvested by centrifugation, and lysed by sonification. The soluble protein was used for purification with the Glutathione Separose 4B column (Amershambiosciences, USA, http://www.amershambiosciences .com).

Analysis of reaction products The reaction mixture for UDP-glycosyltransferase contained 25 µg of the purified RUGT-5, 10 mM KH2 PO4 (pH 7.4), 5 mM MgCl2 , 500 µM UDP-glucose, and 70 µM of substrates. The flavonoids were purchased from Indofinechemicals (New Jersey, USA, http://www.indofinechemical.com). The reaction mixture was incubated at 37◦ C for 30 min and extracted twice with ethylacetate. The ethylacetate was then evaporated completely. Metabolites were analysed using an Agilent HPLC (series 100, USA, http://www.agilent.com) equipped with a photo diode array (PDA) detector and a Waters Symmetry C18 column (3.5 µm particle size, 4.6 mm × 250 mm, Milford, MA, USA, http://www.waters.com). For the analytical scale, the mobile phase consisted of 0.1% formic acid (pH 3.0) and was programmed as follows: 20% acetonitrile at 0 min, 40% acetonitrile at 10 min, 70% acetonitrile at 20 min, 90% acetonitrile at 30 min, 90% acetonitrile at 35 min and 20% acetonitrile at 40 min. The flow rate was 1 ml/min and UV detection was performed at 270 nm. Quantification of the metabolites and the parent material was monitored using HPLC in duplicate experiments. Several different concentrations of each substrate were analysed with HPLC, and the resulting value was used as a standard for the analysis of remaining reaction product after enzymatic conversion of substrates. One unit of enzyme was defined as the amount of enzyme that produced 1 pmole of product in a minute. Results and discussion Cloning and expression of RUGT-5 The rice genome was searched with the plant secondary product UGT consensus sequence (called PSPG motif; Hughes and Hughes 1994), WAPQVELAAHPAVGCFVTHCGWNSTLSESISAGVPMVAWPFFADQ. In addition, the CAZY database was examined and 193 rice Family 1 GTs were found. Among these, we cloned 30 UGTs from rice that showed a high degree of homology with flavonoid glycosyltransferases. Among the 30 UGT genes studied, one UGT (RUGT-5; GenBank accession number XM 463383) was further characterized. RUGT-5 was cloned by RT-PCR and sequenced. RUGT-5 consists of a 1,485 bp open reading frame encoding the 53.5 kDa protein. The predicted protein sequence had 40–42% identity with glycosyltransferases from Catharanthus roseus, Nicotiana tabacum and Dorotheanthus bellidiformis. A phylogenetic tree of flavonoid UGTs showed three groups; the first group contained flavonoid 3-O-glycosyltransferase, the second group consisted of flavonoid 5-O-glucosyltransfrerase and the third group was composed of UGTs displaying diverse regioselectivity (Fig. 1). RUGT-5 was most similar to the third group, with strong correlation for its regioselectivity (see below). In order to determine the substrate, RUGT-5 was sub-cloned into a pGEX E. coli expression vector. The recombinant

743 Zea 3GT Hordeum 3GT Gentiana 3GT Petunia 3GT Gentiana 3'GT

Vigna 3GaT

Forsythia hi 3GT T Perilla 3GT Aralia ia 3GaT Petunia 3GaT

Dorotheanthus 5GT

Vitis 3GT Malus 3GT Citrus GT

Scutellaria 7GT Brassica a GT RUGT-5 Iris 5GT Solanum GT

Medicago di GT

Nicotiana a GT

Verbena 5GT

Petunia 5GT 0.1 Perilla 5GT

Torenia 5GT

Fig. 1 Phylogenetic tree of UDP-dependent glycosyltransferase. 3-O-Glycosyltransferases form one group, 5-O-glycosyltransferases form second group, and UGTs showing diverse regioselectivity form third group. GenBank accession numbers of the UGTs: Medicago GT(AY747627), Solanum GT(STU82367), Scutellaria 7GT(AB031274), Dorotheanthus 5GT(Y18871), Gentiana 3 GT (AB076697), Hordeum 3GT(X15694), Zea 3GT(AY167672),

Vigna 3GaT(AB009370), Petunia 3GT(AB027454), Gentiana 3GT(D85186), Forsythia 3GT(AF127218), Perilla 3GT (AB002818), Aralia 3GaT(AB103471), Petunia 3GaT(AF165148), Vitis 3GT(AB047092), Malus 3GT(AF117267), Citrus GT(AB 033758), Brassica GT(A62529), Iris 5GT(AB113664), Nicotiana GT(AB072919), Petunia 5GT(AB027455), Torenia 5GT(AB 076698), Perilla 5GT(AB013596) and Verbena 5GT(AB013598)

RUGT-5 was detected in the soluble fraction of E. coli lysate and purified as a gluthatione S-transferase (GST) fusion protein (Fig. 2). The resulting molecular weight of the purified recombinant RUGT-5 was approximately 79.5 kDa, which is consistent with the combined molecular weights of GST (26 kDa) and RUGT-5. Expression of RUGT-5 was investigated using real-time quantitative reverse transcriptase-polymerase chain reaction by methods described in Kim et al. (2005). RUGT5 was expressed in seed, root, stem and leaf tissue, although the expression in leaves was found to be approximately 3-fold higher than in other tissues (data not shown). Flavonoids profiling of different rice tissues also showed that leaves contained higher levels of flavonoids than roots (unpublished data), consistent with levels observed in Arabidopsis thaliana (Tohge et al. 2005).

showed that all substrates tested, except catechol and caffeic acid, produced new reaction product(s). This indicated that RUGT-5 might transfer a glucose group to flavonoids as well as to isoflavonoids. According to Meβner et al.

Determination of the substrate of RUGT-5 Based on protein sequence homology, RUGT-5 was assumed to use phenylpropanoid compounds as substrates. Six flavonoids (apigenin, eriodictyol, kaempferol, luteolin, naringenin and quercetin), two isoflavonoids (daidzein and genistein) and three other phenolic compounds (catechol, caffeic acid and esculetin) were tested as possible substrates for the recombinant RUGT-5 protein. The reaction products were analysed by either TLC or HPLC. TLC analysis

Fig. 2 Expression and purification of the recombinant RUGT-5. M—molecular weight marker, 1—uninduced E. coli lysate, 2— induced E. coli total lysate, 3—soluble fraction of induced E. coli lysate, 4—the purified recombinant RUGT-5

P1

140 120 100 80 60 40 20 0

P2 P3

0

Absorbance (mAU)

5

10

15 B

100 Absorbance (mAU)

S1

175 150 125 100 75 50 25 0

80 60 40

P1 S1

20 0 240

0

5

280 320 360 400 Wave length (nm)

10

15 C

P5

175 150 125 100 75 50 25 0

P4

0

5

10

15 D

S2

120 100 80 60 40 20 0

100 80 60 40

P5 S2

20 0 240

0

5

175 150 125 100 75 50 25 0

280 320 360 400 Wave length (nm)

10

15 E

P6

0

5

10

15 F

S3 200 150 100

100 80 60 40

50

P6 S3

20 0 240

280 320 360 Wave length (nm)

400

0 0

5

(2003), Arabidopsis glucosyltransferases that showed activity towards endogenous flavonoids also transferred a glucose residue into xenobiotics 2,4,5-trichlorophenol (TCP). HPLC analysis of the reaction products revealed at least two peaks different from substrates. For example, reaction products of kaempferol showed three peaks while those of luteolin, quercetin and naringenin showed two (Fig. 3). LC/MS analysis of the reaction products from each flavonoids demonstrated that molecular weight was increased by 164 Da compared to the weight of the substrate, which corresponded with the mass of glucose. These results indicated that one glucose molecule was transferred into each substrate. Regiospecificity of RUGT-5 was examined by comparison of HPLC retention time and UV spectra of reaction products with the corresponding authentic glycosylated compounds. In addition, the hypsochromic shift of UVabsorbances between substrates and reaction products was examined due to the observation of a hypsochromic shift when the glycosylated positions are at either 3 or 4 in the flavone or flavonol. In contrast, glycoslation at the 7hydroxyl group does not demonstrate a hypsochromic shift (Vogt et al. 1997; Kramer et al. 2003). The reaction products of kaempferol displayed three peaks (P1–P3 in Fig. 3A). The observed retention time and the UV-spectrum of P1 that appeared as the major peak were indistinguishable from those of the authentic 3-O-kaempferol glucoside, indicating that P1 is a 3-Okaempferol glucoside. P2 showed a hypsochromic shift, while P3 did not (Table 1). This suggests that P2 is likely to be a 4 -O-glucoside of kaempferol and P3 was likely to be a 7-O-glucoside of kaempferol. RUGT-5 yielded two products with luteolin (P4 and P5 in Fig. 3C). P5 had a retention time and UV-spectrum similar to those of the luteolin 4 -O-glucoside and P4 was tentatively assigned Table 1 HPLC retention time, absorbance maximum and structure assignment of RUGT-5 reaction products

P7

Absorbance (mAU)

Absorbance (mAU)

A

Absorbance (mAU)

Absorbance (mAU)

744

10

15

Retention time (min)

Fig. 3 HPLC profile of reaction products with RUGT-5. A Kaempferol reaction products. B Authentic kaempferol-3-Oglucoside. C Luteolin reaction products. D Authentic luteolin-4 O-glucoside. E Apigenin reaction products. F Authentic apigenin -7-O-glucoside. Right insets in B, D and F are UV spectra for authentic compounds and major reaction products

Compounds Retention time (min)

UV (nm) Structure assignment

Kaempferol P1a P2b P3b Luteolin P4a P5a Apigenin P6a P7b Quercetin Minorb Majora

266, 366 266, 348 266, 366 266, 362 256, 348 256, 348 268, 338 268, 338 268, 338 268, 324 256, 370 256, 356 254, 366

a

13.9 6.7 7.2 7.7 11.0 5.8 7.2 13.9 7.4 7.7 11.0 5.7 7.3

Kaempferol aglycon Kaempferol-3-O-glucoside Kaempferol-7-O-glucoside Kaempferol-4 -O-glucoside Luteolin aglycon Luteolin-7-O-glucoside Luteolin-4 -O-glucoside Apigenin aglycon Apigenin-7-O-glucoside Apigenin-4 -O-glucoside Quercetin aglycon Quecetin-3-O-glucoside Quercetin-4 -O-glucoside

These reaction products were assigned according to comparison of HPLC retention time and UV spectra with authentic compounds b These reaction products were assigned according to the hypsochromic shift

745 Table 2 Substrate specificity of the purified recombinant protein RUGT-5

Substrate

Km (µM)

Vmax (pkat/mg)

Vmax /Km

Kaempferol Apigenin Genistein

239.5 327 120.7

1666.7 2000 733.3

6.96 6.11 6.07

(pkat mg−l

µM−l)

Kcat /Km

(µM−l

s−l)

0.37 0.32 0.32

Enzyme assays were carried out using 25 µg of the purified RUGT-5, 10–200 µM of each substrates and 500 µM of UDP-glucose

as a luteolin 7-O-glucoside with a hypsochromic shift (Table 1). In the case of quercetin, two reaction products were also produced: the major product was determined to be a quercetin 4 -O-glucoside after comparison with the authentic compound (data not shown) and the minor product was likely a quercetin-3-O-glucoside based on hypsochromic shift (Table 1). Likewise, the two reaction products of apigenin were determined to be apigenin 7-Oglucoside (P6) and apigenin 4 -O-glucoside (P7) (Fig. 3E and 3F; Table 2). Two reaction products from naringenin were also observed (data not shown). The major reaction product was labelled naringenin 7-O-glucoside, based on comparison of the retention time and UV spectra with the authentic compound, and the minor product labelled narigenin 4 -O-glucoside because narigenin has three hydroxyl groups at C-5, C-7 and C-4 and narignenin 4 -O-glucoside is common in nature. In summary, RUGT-5 transferred a glucose molecule to the hydroxyl group of either C-3, C-7 or C-4 . When both C-3 and C-4 hydroxyl groups were present as seen in quercetin and luteolin, 4 -O-glucoside was a major product of RUGT-5. In case where only the C-4 hydroxyl group was present (as in kaempferol, naringenin and apigenein), the major product was determined by the presence of C-3 hydroxyl group. That is, when the C-3 hydroxyl group was present, the C-3 glycosylated product appeared as the major product, while in the absence of a C-3 hydroxyl group, the C-7 glycosylated product was the major product. UGTs from plants also displayed broad regioselectivity. UGT73G1 from Allium cepa transferred glucose(s) into hydroxyl groups at the C-3, C-7 and C-4 positions of the quercetin (Kramer et al. 2003). 5-GT from Dorotheanthus bellidiformis glycosylated quercetin at either the 4 -hydroxyl or the 7 -hydroxyl group, at a ratio of 3 to 1, respectively (Vogt et al. 1997). The kinetic parameters Km and Vmax for apigenin, genistein and kaempferol were determined using Lineweaver– Burk plots (Table 2). According to Kcat /Km ratio that reflects the enzyme catalytic efficiency, RUGT-5 used kaempferol most efficiently although a high enzymatic affinity towards genistein was observed. The relative activity of RUGT-5 was examined with eight different (iso) flavonoids. As shown in Table 3, the best substrates were keampferol and apigenin, followed by and genistein. Several glycosylated phenolic compounds such as flavonoids and phenylpropanoids were also found in rice. Apigenin, kaempferol and luteolin were most likely to be present, based on UV spectrum and HPLC retention time (unpublished data). These flavonoids exist in glycosylated forms even though the dominant forms of sugar observed

Table 3

Relative activity of RUGT-5 towards several substrates

Compound

Conversion rate (%) Structure

Kaempferol 100a

Apigenin

95

Genistein

94

Luteolin

84

Eriodictyol

80

Quercetin

70

Naringenin

58

a

100% is equivalent to 500 pkat/mg 100 µM of substrate was used and the reaction mixture was incubated at 37◦ C for 20 min b

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in rice several glycosylated flavonoids are not known. According to in vitro results and the known composition of flavonoids in rice, the in vivo substrate of RUGT-5 is likely to be kaempferol. Thus, it is likely that RUGT-5 has a role in storing kaempferol after conversion to its glycoside. Currently, several UGTs from plants have been cloned and characterized. With the completion of the genome project for Arabidopsis thaliana, UGTs have been studied extensively due to the availability of several mutant lines. In contrast, rice whose genome project have been completed is an important model crop plant whose UGTs have not been studied. Therefore, RUGT-5 is the first UGT for rice to have a characterized function. The glycosylation pattern of flavonoids has an effect on bioavailability (Aziz et al. 1998), but chemical glycosylation is difficult and expensive (Arend et al. 2001). Due to its broad range of substrates and low regioselectivity, RUGT5 may be an attractive enzyme for engineering flavonoid diversity. It will also be interesting to determine if RUGT-5 can glycosylate other structurally-related compounds, including antibiotics. Acknowledgements This work was supported by a grant of Biogreen 21 Program, Rural Development Administration, Republic of Korea and in part by KRF2004-F00019 and the R&D Program for NBT Fusion Strategy of Advanced Technologies (Korea Ministry of Commerce, Industry and Energy).

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