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.
Highlights
The formation of isoflavonoid scaffold is conserved in legumes (Veitch, 2009; Ferreyra et al, 2012; Cheynier et al, 2013), the isoflavonoid composition and content are largely different between the species due to a variety of modifications on the compound backbone by specific modifying enzymes, conferring their unique health benefits for human
Glycosylation reactions are catalyzed by uridine diphosphate (UDP)-sugar glycosyltransferases (UGTs) and UGTs acting on plant natural chemicals usually belong to family 1 UGTs, which are characterized by a plant UGT signature, the plant secondary product glycosyltransferase consensus sequence (PSPG) motif (Vogt and Jones, 2000)
UGTs exist as very large families, for example, over 150 different family 1 UGT genes were identified in Medicago truncatula (Modolo et al, 2007), and 117 putative family 1 UGT genes were found in Pueraria lobata (Wang et al, 2015)
Summary
The formation of isoflavonoid scaffold is conserved in legumes (Veitch, 2009; Ferreyra et al, 2012; Cheynier et al, 2013), the isoflavonoid composition and content are largely different between the species due to a variety of modifications on the compound backbone by specific modifying enzymes, conferring their unique health benefits for human. Such enzymatic modifications, including hydroxylation, glycosylation, methylation, and acylation, change the. More than 100 putative UGT genes were predicted from the P. lobata species, only three UGTs were functionally characterized in P. lobata (He et al, 2011; Li et al, 2014)
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