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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

Phylogenetic tree analysis of PlUGT2 with other known UGTs. Twenty two identified P. lobata family 1 UGTs were aligned with other plant UGTs whose functions have been characterized, including seven soybean UGTs and three kudzu UGTs. The tree was constructed from a MEGA 6.0 program using a neighbor-joining method (with 1000 bootstrap replications). Names and accession numbers of UGTs used for the alignment are as follows. AmC4’GT (Antirrhinum majus UDP-glucose: chalcone 4′-O-glucosyltransferase, AB198665); BMGT1 (Bacopa monnieri genistein 4′-O-glucosyltransferase, UGT74W1, ACM09993); GmUGT2 (Glycine max UGT2, AB904891); GmUGT3 (G. max UGT3, AB904892); GmUGT4 (G. max UGT4, AB904893); GmUGT7 (G. max UGT7, AB904894); GmUGT8 (G. max UGT8, AB904895); GmUGT9 (G. max UGT9, AB904896); HpUGT90A7 (Pilosella officinarum flavonoid glucosyltransferases, ACB56926); HpUGT72B11 (P. officinarum coniferyl-alcohol glucosyltransferase, ACB56923); LvC4′GT (Linaria vulgaris UDP-glucose: chalcone 4′-O-glucosyltransferase, BAE48240); OsCGT (Oryza sativa flavoniod C-glucosyltransferase, CAQ77160). PlUGT1 (P. lobata isoflavone 7-O-glucosyltransferase 1, KC473565); PlUGT2 (P. lobata isoflavone 4′,7-O-glucosyltransferase, KU311040); PlUGT13 (P. lobata isoflavone 7-O-glucosyltransferase 13, KC473566); PlUGT15 (P. lobata glucosyltransferase 15, KU311041); PlUGT18 (P. lobata glucosyltransferase 18, KC473567); GT04F14 (P. lobata isoflavone 7-O-glucosyltransferase, HQ219042), GT14A05 (P. lobata flavone glucosyltransferase, HQ219047). The sequences of the other 20 P. lobata glucosyltransferases were submitted to the GenBank database with the accession nos. KU317800–KU317819.
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Figure 3: Phylogenetic tree analysis of PlUGT2 with other known UGTs. Twenty two identified P. lobata family 1 UGTs were aligned with other plant UGTs whose functions have been characterized, including seven soybean UGTs and three kudzu UGTs. The tree was constructed from a MEGA 6.0 program using a neighbor-joining method (with 1000 bootstrap replications). Names and accession numbers of UGTs used for the alignment are as follows. AmC4’GT (Antirrhinum majus UDP-glucose: chalcone 4′-O-glucosyltransferase, AB198665); BMGT1 (Bacopa monnieri genistein 4′-O-glucosyltransferase, UGT74W1, ACM09993); GmUGT2 (Glycine max UGT2, AB904891); GmUGT3 (G. max UGT3, AB904892); GmUGT4 (G. max UGT4, AB904893); GmUGT7 (G. max UGT7, AB904894); GmUGT8 (G. max UGT8, AB904895); GmUGT9 (G. max UGT9, AB904896); HpUGT90A7 (Pilosella officinarum flavonoid glucosyltransferases, ACB56926); HpUGT72B11 (P. officinarum coniferyl-alcohol glucosyltransferase, ACB56923); LvC4′GT (Linaria vulgaris UDP-glucose: chalcone 4′-O-glucosyltransferase, BAE48240); OsCGT (Oryza sativa flavoniod C-glucosyltransferase, CAQ77160). PlUGT1 (P. lobata isoflavone 7-O-glucosyltransferase 1, KC473565); PlUGT2 (P. lobata isoflavone 4′,7-O-glucosyltransferase, KU311040); PlUGT13 (P. lobata isoflavone 7-O-glucosyltransferase 13, KC473566); PlUGT15 (P. lobata glucosyltransferase 15, KU311041); PlUGT18 (P. lobata glucosyltransferase 18, KC473567); GT04F14 (P. lobata isoflavone 7-O-glucosyltransferase, HQ219042), GT14A05 (P. lobata flavone glucosyltransferase, HQ219047). The sequences of the other 20 P. lobata glucosyltransferases were submitted to the GenBank database with the accession nos. KU317800–KU317819.

Mentions: By the use of RNA-sequencing technology, we previously reported 22 P. lobata family 1 PlUGTs that are preferentially expressed in its roots over leaves (Wang et al., 2015). For the isolation of 4′-O-UGTs from P. lobata, these 22 UGTs were phylogenetically analyzed with other plant UGTs whose functions have been characterized, which include seven soybean UGTs (Funaki et al., 2015) and three kudzu UGTs (PlUGT1, PlUGT13, and GT04F14; He et al., 2011; Li et al., 2014). The result showed that two putative PlUGTs (named as PlUGT2 and PlUGT15) were clustered into the same group with the soybean GmUGTs and PlUGT1 (group I), while the remaining 20 PlUGTs formed another group (group II; Figure 3). Furthermore, PlUGT2 and PlUGT15 were found to be scattered into two separate clades in the group I. PlUGT15 together with PlUGT1 displayed relatively higher homology to GmUGTs of subgroup A, whereas PlUGT2 showed a closer relationship with subgroup B members (Funaki et al., 2015). In subgroup A, GmUGT members (GmUGT3, GmUGT4, and GmUGT9) and PlUGT1 were characterized to be isoflavone specific 7-O-UGTs. The deduced amino acid sequence of PlUGT15 shared above 90% sequence identity with PlUGT1, and it indeed showed the same activity as PlUGT1 (data not shown). In subgroup B, PlUGT2 was in the same branch with GmUGT1 and GmUGT7, and had 82% amino acid sequence similarity to GmUGT1. It was reported that GmUGT1 and GmUGT7 not only efficiently glycosylated isoflavone aglycones at the 7-hydroxy group, but also exhibited considerable 4′-O-glucosylation activities toward some flavonoids. On the other hand, in the group II, PlUGT18 clustered with BMGT1 (UGT74W1) which specifically glucosylates the 4′-O-position of genistein (Ruby et al., 2014). The biochemical function of PlUGT18 was examined by our previous research and no activities toward any of the isoflavones were found (Li et al., 2014). Thus, taken together, the phylogenic tree analysis here made PlUGT2 an interesting candidate for functional characterization and testing of a possible role in the 4′-O-glucosylations of P. lobata isoflavonoids.


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)

Phylogenetic tree analysis of PlUGT2 with other known UGTs. Twenty two identified P. lobata family 1 UGTs were aligned with other plant UGTs whose functions have been characterized, including seven soybean UGTs and three kudzu UGTs. The tree was constructed from a MEGA 6.0 program using a neighbor-joining method (with 1000 bootstrap replications). Names and accession numbers of UGTs used for the alignment are as follows. AmC4’GT (Antirrhinum majus UDP-glucose: chalcone 4′-O-glucosyltransferase, AB198665); BMGT1 (Bacopa monnieri genistein 4′-O-glucosyltransferase, UGT74W1, ACM09993); GmUGT2 (Glycine max UGT2, AB904891); GmUGT3 (G. max UGT3, AB904892); GmUGT4 (G. max UGT4, AB904893); GmUGT7 (G. max UGT7, AB904894); GmUGT8 (G. max UGT8, AB904895); GmUGT9 (G. max UGT9, AB904896); HpUGT90A7 (Pilosella officinarum flavonoid glucosyltransferases, ACB56926); HpUGT72B11 (P. officinarum coniferyl-alcohol glucosyltransferase, ACB56923); LvC4′GT (Linaria vulgaris UDP-glucose: chalcone 4′-O-glucosyltransferase, BAE48240); OsCGT (Oryza sativa flavoniod C-glucosyltransferase, CAQ77160). PlUGT1 (P. lobata isoflavone 7-O-glucosyltransferase 1, KC473565); PlUGT2 (P. lobata isoflavone 4′,7-O-glucosyltransferase, KU311040); PlUGT13 (P. lobata isoflavone 7-O-glucosyltransferase 13, KC473566); PlUGT15 (P. lobata glucosyltransferase 15, KU311041); PlUGT18 (P. lobata glucosyltransferase 18, KC473567); GT04F14 (P. lobata isoflavone 7-O-glucosyltransferase, HQ219042), GT14A05 (P. lobata flavone glucosyltransferase, HQ219047). The sequences of the other 20 P. lobata glucosyltransferases were submitted to the GenBank database with the accession nos. KU317800–KU317819.
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Figure 3: Phylogenetic tree analysis of PlUGT2 with other known UGTs. Twenty two identified P. lobata family 1 UGTs were aligned with other plant UGTs whose functions have been characterized, including seven soybean UGTs and three kudzu UGTs. The tree was constructed from a MEGA 6.0 program using a neighbor-joining method (with 1000 bootstrap replications). Names and accession numbers of UGTs used for the alignment are as follows. AmC4’GT (Antirrhinum majus UDP-glucose: chalcone 4′-O-glucosyltransferase, AB198665); BMGT1 (Bacopa monnieri genistein 4′-O-glucosyltransferase, UGT74W1, ACM09993); GmUGT2 (Glycine max UGT2, AB904891); GmUGT3 (G. max UGT3, AB904892); GmUGT4 (G. max UGT4, AB904893); GmUGT7 (G. max UGT7, AB904894); GmUGT8 (G. max UGT8, AB904895); GmUGT9 (G. max UGT9, AB904896); HpUGT90A7 (Pilosella officinarum flavonoid glucosyltransferases, ACB56926); HpUGT72B11 (P. officinarum coniferyl-alcohol glucosyltransferase, ACB56923); LvC4′GT (Linaria vulgaris UDP-glucose: chalcone 4′-O-glucosyltransferase, BAE48240); OsCGT (Oryza sativa flavoniod C-glucosyltransferase, CAQ77160). PlUGT1 (P. lobata isoflavone 7-O-glucosyltransferase 1, KC473565); PlUGT2 (P. lobata isoflavone 4′,7-O-glucosyltransferase, KU311040); PlUGT13 (P. lobata isoflavone 7-O-glucosyltransferase 13, KC473566); PlUGT15 (P. lobata glucosyltransferase 15, KU311041); PlUGT18 (P. lobata glucosyltransferase 18, KC473567); GT04F14 (P. lobata isoflavone 7-O-glucosyltransferase, HQ219042), GT14A05 (P. lobata flavone glucosyltransferase, HQ219047). The sequences of the other 20 P. lobata glucosyltransferases were submitted to the GenBank database with the accession nos. KU317800–KU317819.
Mentions: By the use of RNA-sequencing technology, we previously reported 22 P. lobata family 1 PlUGTs that are preferentially expressed in its roots over leaves (Wang et al., 2015). For the isolation of 4′-O-UGTs from P. lobata, these 22 UGTs were phylogenetically analyzed with other plant UGTs whose functions have been characterized, which include seven soybean UGTs (Funaki et al., 2015) and three kudzu UGTs (PlUGT1, PlUGT13, and GT04F14; He et al., 2011; Li et al., 2014). The result showed that two putative PlUGTs (named as PlUGT2 and PlUGT15) were clustered into the same group with the soybean GmUGTs and PlUGT1 (group I), while the remaining 20 PlUGTs formed another group (group II; Figure 3). Furthermore, PlUGT2 and PlUGT15 were found to be scattered into two separate clades in the group I. PlUGT15 together with PlUGT1 displayed relatively higher homology to GmUGTs of subgroup A, whereas PlUGT2 showed a closer relationship with subgroup B members (Funaki et al., 2015). In subgroup A, GmUGT members (GmUGT3, GmUGT4, and GmUGT9) and PlUGT1 were characterized to be isoflavone specific 7-O-UGTs. The deduced amino acid sequence of PlUGT15 shared above 90% sequence identity with PlUGT1, and it indeed showed the same activity as PlUGT1 (data not shown). In subgroup B, PlUGT2 was in the same branch with GmUGT1 and GmUGT7, and had 82% amino acid sequence similarity to GmUGT1. It was reported that GmUGT1 and GmUGT7 not only efficiently glycosylated isoflavone aglycones at the 7-hydroxy group, but also exhibited considerable 4′-O-glucosylation activities toward some flavonoids. On the other hand, in the group II, PlUGT18 clustered with BMGT1 (UGT74W1) which specifically glucosylates the 4′-O-position of genistein (Ruby et al., 2014). The biochemical function of PlUGT18 was examined by our previous research and no activities toward any of the isoflavones were found (Li et al., 2014). Thus, taken together, the phylogenic tree analysis here made PlUGT2 an interesting candidate for functional characterization and testing of a possible role in the 4′-O-glucosylations of P. lobata isoflavonoids.

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