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Molecular Cloning and Functional Characterization of a Novel (Iso)flavone 4',7-O-diglucoside Glucosyltransferase from Pueraria lobata.

Wang X, Fan R, Li J, Li C, Zhang Y - Front Plant Sci (2016)

Bottom Line: Pueraria lobata roots accumulate a rich source of isoflavonoid glycosides, including 7-O- and 4'-O-mono-glucosides, and 4',7-O-diglucosides, which have numerous human health benefits.Real-time PCR analysis showed that PlUGT2 is preferentially transcribed in roots relative to other organs of P. lobata, which is coincident with the accumulation pattern of 4'-O-glucosides and 4',7-O-diglucosides in P. lobata.The identification of PlUGT2 would help to decipher the P. lobata isoflavonoid glucosylations in vivo and may provide a useful enzyme catalyst for an efficient biotransformation of isoflavones or other natural products for food or pharmacological purposes.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences Wuhan, China.

ABSTRACT
Pueraria lobata roots accumulate a rich source of isoflavonoid glycosides, including 7-O- and 4'-O-mono-glucosides, and 4',7-O-diglucosides, which have numerous human health benefits. Although, isoflavonoid 7-O-glucosyltranferases (7-O-UGTs) have been well-characterized at molecular levels in legume plants, genes, or enzymes that are required for isoflavonoid 4'-O- and 4',7-O-glucosylation have not been identified in P. lobata to date. Especially for the 4',7-O-di-glucosylations, the genetic control for this tailing process has never been elucidated from any plant species. Through transcriptome mining, we describe here the identification and characterization of a novel UGT (designated PlUGT2) governing the isoflavonoid 4',7-O-di-glucosylations in P. lobata. Biochemical roles of PlUGT2 were assessed by in vitro assays with PlUGT2 protein produced in Escherichia coli and analyzed for its qualitative substrate specificity. PlUGT2 was active with various (iso)flavonoid acceptors, catalyzing consecutive glucosylation activities at their O-4' and O-7 positions. PlUGT2 was most active with genistein, a general isoflavone in legume plants. Real-time PCR analysis showed that PlUGT2 is preferentially transcribed in roots relative to other organs of P. lobata, which is coincident with the accumulation pattern of 4'-O-glucosides and 4',7-O-diglucosides in P. lobata. The identification of PlUGT2 would help to decipher the P. lobata isoflavonoid glucosylations in vivo and may provide a useful enzyme catalyst for an efficient biotransformation of isoflavones or other natural products for food or pharmacological purposes.

No MeSH data available.


Related in: MedlinePlus

High-performance liquid chromatography (HPLC) analysis of the products from the in vitro reactions of the recombinant PlUGT2 with genitein (A), liquiritigenin (B), and daidzein (C). PlUGT2 was able to converted these (iso)flavone aglycones to yield their respective 4′,7-O-diglucosides (peaks 1, 4, 7), 7-O-mono-glucosides (peaks 2, 5, 8), and 4′-O-mono-glucosides (peaks 3, 6, 9). (-) Indicates control reactions without the addition of PlUGT2. Peak 1, genitein 4′,7-O-diglucoside; peak 2, genitin (genitein 7-O-glucoside); peak 3, sophoricoside (genitein 4′-O-glucoside); peak 4, liquiritigenin 4′,7-O-diglucoside; peak 5, neoliquiritin (liquiritigenin 7-O-glucoside); peak 6, liquiritin (liquiritigenin 4′-O-glucoside); peak 7, daidzein 4′,7-O-diglucoside; peak 8, daidzin (daidzein 7-O-glucoside); peak 9, daidzein 4′-O-glucoside. The mass spectra of all the reaction products (peaks 1–9) were shown in Supplementary Figure S3, and their chemical structures are listed in the Figure 1.
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Figure 4: High-performance liquid chromatography (HPLC) analysis of the products from the in vitro reactions of the recombinant PlUGT2 with genitein (A), liquiritigenin (B), and daidzein (C). PlUGT2 was able to converted these (iso)flavone aglycones to yield their respective 4′,7-O-diglucosides (peaks 1, 4, 7), 7-O-mono-glucosides (peaks 2, 5, 8), and 4′-O-mono-glucosides (peaks 3, 6, 9). (-) Indicates control reactions without the addition of PlUGT2. Peak 1, genitein 4′,7-O-diglucoside; peak 2, genitin (genitein 7-O-glucoside); peak 3, sophoricoside (genitein 4′-O-glucoside); peak 4, liquiritigenin 4′,7-O-diglucoside; peak 5, neoliquiritin (liquiritigenin 7-O-glucoside); peak 6, liquiritin (liquiritigenin 4′-O-glucoside); peak 7, daidzein 4′,7-O-diglucoside; peak 8, daidzin (daidzein 7-O-glucoside); peak 9, daidzein 4′-O-glucoside. The mass spectra of all the reaction products (peaks 1–9) were shown in Supplementary Figure S3, and their chemical structures are listed in the Figure 1.

Mentions: In the in vitro enzyme assays, PlUGT2 was tested against various substrates and found to be active with some (iso)flavonoids using UDP-glucose as the sugar donor (Table 1). Among the active substrates, genistein was the best acceptor (100% relative activity, 136.96 ± 4.24 nmol mg protein-1 min-1) followed by formononetin (83.48% relative activity, 114.33 ± 4.35 nmol mg protein-1 min-1), daidzein (38.93% relative activity, 53.32 ± 1.95 nmol mg protein-1 min-1), liquiritigenin (11.83% relative activity, 16.20 ± 3.72 nmol mg protein-1 min-1), and naringenin (5.59% relative activity, 7.66 ± 1.32 nmol mg protein-1 min-1). Other (iso)flavonoids were poor substrates of PlUGT2 with their relative activities varying from 0.08 to 1.91% (Table 1). When genistein was used as the substrate, three new products (peaks 1–3) were formed in comparison with the control reaction (Figure 4A). By examining the retention times and mass spectra with chemical standards, the peak 2 and peak 3 could be identified as genistin (genistein 7-O-glucoside) and genistein 4′-O-glucoside (namely sophoricoside), respectively (Supplementary Figures S3A,B,G,H). This data suggested that PlUGT2 catalyzes either 7-O- or 4′-O-glucosylation activity toward genistein. The peak 2 and peak 3 had a molecular ion of m/z+ 433 while the molecular ion for the peak 1 was 595 (Supplementary Figure S3F), indicating that the peak 1 could be genistein diglucoside in which a glucose group might be attached to both O-4′ and O-7 positions. To test this hypothesis, we attempted in vitro assays using genistin (genistein 7-O-glucoside) and sophoricoside (genistein 4′-O-glucoside) as the substrates. Apparently, the product peak 1 could be detected in both reactions (Supplementary Figures S4A,B), confirming that the peak 1 is genistein 4′,7-O-diglucoside. Thus, this suggested that PlUGT2 could transfer a glucose group to genistein at O-4′ or O-7 position and further convert the mono-glucosides to genistein diglucoside. It should be mentioned that the specific activity of PlUGT2 toward genistein mono-glucosides (0.15 ± 0.07 nmol mg protein-1 min-1 for genistin, 2.62 ± 0.16 nmol mg protein-1 min-1 for sophoricoside) was much lower than that toward its aglycone (136.96 ± 4.24 nmol mg protein-1 min-1; Table 1). Similarly, PlUGT2 transferred a glucose group to liquiritigenin at O-7 position to yield neoliquiritin (peak 5), at O-4′ position to yield liquiritin (peak 6), and both O-4′ and O-7 positions to produce the 4′,7-O-diglucoside of liquiritigenin (peak 4; Figure 4B). The peak 4 was postulated as the 4′,7-O-diglucoside of liquiritigenin based on its mass spectrum (Supplementary Figure S3I) and the results from the in vitro assays of PlUGT2 with neoliquiritin or liquiritin (Supplementary Figure S4C). The identities of the peak 5 and peak 6 were determined by comparing retention times and mass spectrums with their corresponding authentic chemicals (Figure 4B, Supplementary Figures S3C,D,J,K). The similar activity was also found in the reactions of PlUGT2 with daidzein (Figure 4C) and naringenin (Supplementary Figure S4D), forming the reaction product peaks 7–12. Except for the peak 8 (Supplementary Figures S3E,M), the authentic chemicals corresponding to the other peaks were not available. However, based on their mass spectrums (Supplementary Figures 3L,N,P–R) and the feature that the earliest eluted glucosides on the HPLC condition of this study were 4′,7-O-diglucosides which were successively followed by 7-O-glucosides and 4′-O-glucosides (Figure 4), the peaks 7–12 were postulated as follows: daidzein 4′,7-O-diglucoside (peak 7), daidzein 7-O-glucoside (peak 8), daidzein 4′-O-glucoside (peak 9), naringenin 4′,7-O-diglucoside (peak 10), naringenin 7-O-glucoside (peak 11), and naringenin 4′-O-glucoside (peak 12). When puerarin and formononetin were used as the substrates, only single product was obtained for each substrate. A single product (peak 13) was formed in the reaction with puerarin (Supplementary Figure S4E). The mass spectrum of the peak 13 indicated that there was a glucose group attached to puerarin (Supplementary Figure S3S). Since there are free hydroxyl groups at the O-4′ and O-7 positions of puerarin molecular, the peak 13 could be either puerarin 7-O-glucoside or puerarin 4′-O-glucoside (Shi et al., 2006). PlUGT2 converted the substrate formononetin to form a single product peak 14 which showed the same retention times and mass spectra with ononin (Supplementary Figures S3T and 4F), suggesting its 7-O-glucosylation activity toward formononetin.


Molecular Cloning and Functional Characterization of a Novel (Iso)flavone 4',7-O-diglucoside Glucosyltransferase from Pueraria lobata.

Wang X, Fan R, Li J, Li C, Zhang Y - Front Plant Sci (2016)

High-performance liquid chromatography (HPLC) analysis of the products from the in vitro reactions of the recombinant PlUGT2 with genitein (A), liquiritigenin (B), and daidzein (C). PlUGT2 was able to converted these (iso)flavone aglycones to yield their respective 4′,7-O-diglucosides (peaks 1, 4, 7), 7-O-mono-glucosides (peaks 2, 5, 8), and 4′-O-mono-glucosides (peaks 3, 6, 9). (-) Indicates control reactions without the addition of PlUGT2. Peak 1, genitein 4′,7-O-diglucoside; peak 2, genitin (genitein 7-O-glucoside); peak 3, sophoricoside (genitein 4′-O-glucoside); peak 4, liquiritigenin 4′,7-O-diglucoside; peak 5, neoliquiritin (liquiritigenin 7-O-glucoside); peak 6, liquiritin (liquiritigenin 4′-O-glucoside); peak 7, daidzein 4′,7-O-diglucoside; peak 8, daidzin (daidzein 7-O-glucoside); peak 9, daidzein 4′-O-glucoside. The mass spectra of all the reaction products (peaks 1–9) were shown in Supplementary Figure S3, and their chemical structures are listed in the Figure 1.
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Figure 4: High-performance liquid chromatography (HPLC) analysis of the products from the in vitro reactions of the recombinant PlUGT2 with genitein (A), liquiritigenin (B), and daidzein (C). PlUGT2 was able to converted these (iso)flavone aglycones to yield their respective 4′,7-O-diglucosides (peaks 1, 4, 7), 7-O-mono-glucosides (peaks 2, 5, 8), and 4′-O-mono-glucosides (peaks 3, 6, 9). (-) Indicates control reactions without the addition of PlUGT2. Peak 1, genitein 4′,7-O-diglucoside; peak 2, genitin (genitein 7-O-glucoside); peak 3, sophoricoside (genitein 4′-O-glucoside); peak 4, liquiritigenin 4′,7-O-diglucoside; peak 5, neoliquiritin (liquiritigenin 7-O-glucoside); peak 6, liquiritin (liquiritigenin 4′-O-glucoside); peak 7, daidzein 4′,7-O-diglucoside; peak 8, daidzin (daidzein 7-O-glucoside); peak 9, daidzein 4′-O-glucoside. The mass spectra of all the reaction products (peaks 1–9) were shown in Supplementary Figure S3, and their chemical structures are listed in the Figure 1.
Mentions: In the in vitro enzyme assays, PlUGT2 was tested against various substrates and found to be active with some (iso)flavonoids using UDP-glucose as the sugar donor (Table 1). Among the active substrates, genistein was the best acceptor (100% relative activity, 136.96 ± 4.24 nmol mg protein-1 min-1) followed by formononetin (83.48% relative activity, 114.33 ± 4.35 nmol mg protein-1 min-1), daidzein (38.93% relative activity, 53.32 ± 1.95 nmol mg protein-1 min-1), liquiritigenin (11.83% relative activity, 16.20 ± 3.72 nmol mg protein-1 min-1), and naringenin (5.59% relative activity, 7.66 ± 1.32 nmol mg protein-1 min-1). Other (iso)flavonoids were poor substrates of PlUGT2 with their relative activities varying from 0.08 to 1.91% (Table 1). When genistein was used as the substrate, three new products (peaks 1–3) were formed in comparison with the control reaction (Figure 4A). By examining the retention times and mass spectra with chemical standards, the peak 2 and peak 3 could be identified as genistin (genistein 7-O-glucoside) and genistein 4′-O-glucoside (namely sophoricoside), respectively (Supplementary Figures S3A,B,G,H). This data suggested that PlUGT2 catalyzes either 7-O- or 4′-O-glucosylation activity toward genistein. The peak 2 and peak 3 had a molecular ion of m/z+ 433 while the molecular ion for the peak 1 was 595 (Supplementary Figure S3F), indicating that the peak 1 could be genistein diglucoside in which a glucose group might be attached to both O-4′ and O-7 positions. To test this hypothesis, we attempted in vitro assays using genistin (genistein 7-O-glucoside) and sophoricoside (genistein 4′-O-glucoside) as the substrates. Apparently, the product peak 1 could be detected in both reactions (Supplementary Figures S4A,B), confirming that the peak 1 is genistein 4′,7-O-diglucoside. Thus, this suggested that PlUGT2 could transfer a glucose group to genistein at O-4′ or O-7 position and further convert the mono-glucosides to genistein diglucoside. It should be mentioned that the specific activity of PlUGT2 toward genistein mono-glucosides (0.15 ± 0.07 nmol mg protein-1 min-1 for genistin, 2.62 ± 0.16 nmol mg protein-1 min-1 for sophoricoside) was much lower than that toward its aglycone (136.96 ± 4.24 nmol mg protein-1 min-1; Table 1). Similarly, PlUGT2 transferred a glucose group to liquiritigenin at O-7 position to yield neoliquiritin (peak 5), at O-4′ position to yield liquiritin (peak 6), and both O-4′ and O-7 positions to produce the 4′,7-O-diglucoside of liquiritigenin (peak 4; Figure 4B). The peak 4 was postulated as the 4′,7-O-diglucoside of liquiritigenin based on its mass spectrum (Supplementary Figure S3I) and the results from the in vitro assays of PlUGT2 with neoliquiritin or liquiritin (Supplementary Figure S4C). The identities of the peak 5 and peak 6 were determined by comparing retention times and mass spectrums with their corresponding authentic chemicals (Figure 4B, Supplementary Figures S3C,D,J,K). The similar activity was also found in the reactions of PlUGT2 with daidzein (Figure 4C) and naringenin (Supplementary Figure S4D), forming the reaction product peaks 7–12. Except for the peak 8 (Supplementary Figures S3E,M), the authentic chemicals corresponding to the other peaks were not available. However, based on their mass spectrums (Supplementary Figures 3L,N,P–R) and the feature that the earliest eluted glucosides on the HPLC condition of this study were 4′,7-O-diglucosides which were successively followed by 7-O-glucosides and 4′-O-glucosides (Figure 4), the peaks 7–12 were postulated as follows: daidzein 4′,7-O-diglucoside (peak 7), daidzein 7-O-glucoside (peak 8), daidzein 4′-O-glucoside (peak 9), naringenin 4′,7-O-diglucoside (peak 10), naringenin 7-O-glucoside (peak 11), and naringenin 4′-O-glucoside (peak 12). When puerarin and formononetin were used as the substrates, only single product was obtained for each substrate. A single product (peak 13) was formed in the reaction with puerarin (Supplementary Figure S4E). The mass spectrum of the peak 13 indicated that there was a glucose group attached to puerarin (Supplementary Figure S3S). Since there are free hydroxyl groups at the O-4′ and O-7 positions of puerarin molecular, the peak 13 could be either puerarin 7-O-glucoside or puerarin 4′-O-glucoside (Shi et al., 2006). PlUGT2 converted the substrate formononetin to form a single product peak 14 which showed the same retention times and mass spectra with ononin (Supplementary Figures S3T and 4F), suggesting its 7-O-glucosylation activity toward formononetin.

Bottom Line: Pueraria lobata roots accumulate a rich source of isoflavonoid glycosides, including 7-O- and 4'-O-mono-glucosides, and 4',7-O-diglucosides, which have numerous human health benefits.Real-time PCR analysis showed that PlUGT2 is preferentially transcribed in roots relative to other organs of P. lobata, which is coincident with the accumulation pattern of 4'-O-glucosides and 4',7-O-diglucosides in P. lobata.The identification of PlUGT2 would help to decipher the P. lobata isoflavonoid glucosylations in vivo and may provide a useful enzyme catalyst for an efficient biotransformation of isoflavones or other natural products for food or pharmacological purposes.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences Wuhan, China.

ABSTRACT
Pueraria lobata roots accumulate a rich source of isoflavonoid glycosides, including 7-O- and 4'-O-mono-glucosides, and 4',7-O-diglucosides, which have numerous human health benefits. Although, isoflavonoid 7-O-glucosyltranferases (7-O-UGTs) have been well-characterized at molecular levels in legume plants, genes, or enzymes that are required for isoflavonoid 4'-O- and 4',7-O-glucosylation have not been identified in P. lobata to date. Especially for the 4',7-O-di-glucosylations, the genetic control for this tailing process has never been elucidated from any plant species. Through transcriptome mining, we describe here the identification and characterization of a novel UGT (designated PlUGT2) governing the isoflavonoid 4',7-O-di-glucosylations in P. lobata. Biochemical roles of PlUGT2 were assessed by in vitro assays with PlUGT2 protein produced in Escherichia coli and analyzed for its qualitative substrate specificity. PlUGT2 was active with various (iso)flavonoid acceptors, catalyzing consecutive glucosylation activities at their O-4' and O-7 positions. PlUGT2 was most active with genistein, a general isoflavone in legume plants. Real-time PCR analysis showed that PlUGT2 is preferentially transcribed in roots relative to other organs of P. lobata, which is coincident with the accumulation pattern of 4'-O-glucosides and 4',7-O-diglucosides in P. lobata. The identification of PlUGT2 would help to decipher the P. lobata isoflavonoid glucosylations in vivo and may provide a useful enzyme catalyst for an efficient biotransformation of isoflavones or other natural products for food or pharmacological purposes.

No MeSH data available.


Related in: MedlinePlus