Functional characterization, structural basis, and regio-selectivity control of a promiscuous flavonoid 7,4′-di-O-glycosyltransferase from Ziziphus jujuba var. spinosa
Abstract:A highly efficient and promiscuous 7,4′-di-O-glycosyltransferase ZjOGT3 was discovered from the medicinal plant Ziziphus jujuba var. spinosa. ZjOGT3 could sequentially catalyse 4′- and 7-O-glycosylation of flavones to produce 7,4′-di-O-glycosides with...
“…5d ). The formation of the respective di- O -glucosides was considerably slower (7.8 mU mg −1 towards apigenin 7,4′-di- O -glucoside; 10.2 mU mg −1 towards luteolin 7,4′-di- O -glucoside, Table 1 ), consistent with previous reports on di- O and di- C -glycosyltransferases capable of catalyzing the serial two-step glycosylation 58 , 59 . Fig.…”
Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions.
“…5d ). The formation of the respective di- O -glucosides was considerably slower (7.8 mU mg −1 towards apigenin 7,4′-di- O -glucoside; 10.2 mU mg −1 towards luteolin 7,4′-di- O -glucoside, Table 1 ), consistent with previous reports on di- O and di- C -glycosyltransferases capable of catalyzing the serial two-step glycosylation 58 , 59 . Fig.…”
Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions.
“…Very limited plant GTs were found capable of sequentially glycosylation of the flavonoids on 7-OH and 4′-OH. Unlike that the recently reported flavonoid 7,4′-di-Oglycosyltransferase (ZjOGT3) initially produces a 4′-O-glucoside, 34 the initial product of UGT74AN3 was determined to be a 7-O-glucoside by time-course experiments (Figure S170). It is noteworthy that UGT74AN3 is not strictly regioselective for the 7-OH and 4′-OH of flavonoids and also exhibits weak 3-OH and 3′-OH activity, e.g., the structures of 2c and 9c were identified as 7,3′-di-O glucoside and 3,7-di-O-glucoside, respectively.…”
Section: Probing the Substrate Promiscuity Of Ugt74an3contrasting
confidence: 60%
“…For example, Sb3GT1 prefers to glycosylate flavonoid compounds and UGT71BD1 prefers to recognize aromatic glycosides. , Moreover, the structural basis for substrate promiscuity of plant UGTs is still unclear. Since the first crystal structure of the plant UGT UGT71G1 was solved, the crystal structures of 27 plant UGTs have been reported. , Among these structures, most of the promiscuous UGTs have been reported in the apo form or as binary complexes with UDP. − Only a few promiscuous UGTs have been reported with bound substrates in the ternary complexes, such as the structures of GgCGT complexed with UDP/phloretin or UDP/nothofagin . The limited structural information about the promiscuous UGT complexes does not provide many mechanistic details of substrate promiscuity.…”
Glycosyltransferases are effective enzymes for glycosylating natural products (NPs), and some of them have the unusual property of being exceedingly promiscuous catalytically toward a range of substrates. UGT74AN3 is a plant glycosyltransferase identified from Catharanthus roseus in our previous work. In this study, we found that UGT74AN3 exhibits high substrate promiscuity toward 78 acceptors and 6 sugar donors and also exhibits N-/S-glycosylation activity toward simple aromatic compounds. The crystal structures of UGT74AN3 in the complex with various NPs were solved. Sugar donor recognition of UGT74AN3 was altered by structure-based mutagenesis, and the T145V mutant shifted its sugar donor preference from UDP-Glc to UDP-Xyl. Structural analysis reveals that a spacious U-shaped hydrophobic binding pocket accounts for the high substrate promiscuity of UGT74AN3. The residues E85 and F193 might serve as gatekeepers of UGT74AN3 to control substrate binding. In addition, a rare substrate binding mode was discovered in the structure of UGT74AN3, and the process of substrate flipping in the pocket was charted by molecular dynamics simulations. Moreover, a cost-effective one-pot system by coupling UGT74AN3 with AtSuSy, a sucrose synthase, was established for in situ generating and recycling UDP-Glc from sucrose and UDP to glycosylate NPs. Our study reveals the structural basis underlying the substrate promiscuity of UGT74AN3 and provides an efficient and economical enzymatic synthesis strategy for producing valuable glycosides for drug discovery.
“…spinosa. ) UGT84 UDP-Glc UDP-Xyl UDP-GlcNAc (22%)* * UDP-Ara p (19%)* * Flavonoids Isoflavonoids Chalcones UDP (8INH) [57] *The sugar donors are listed in order of preference. * * Percentage represents the conversion rate, whereas “weak” represents low catalytic activity.…”
Section: Structural Insights Into Plant Ugtsmentioning
confidence: 99%
“…However, there are exceptions to this model, including the catalytic mechanisms for the Ziziphus jujuba flavonoid 7,4′-di-O-glycosyltransferase ( Zj OGT3) [57] , Trollius chinensis O-/C-glycosyltransferase ( Tc CGT1) [37] , Phytolacca americana polyphenol glycosyltransferase ( Pa GT2) [32] , Zea mays silibinin glucosyltransferase UGT706F8 [13] , and At UGT89C1 [53] . In the O-glycosylation reactions catalyzed by Zj OGT3 and Tc CGT1, spontaneous deprotonation of the catalytic hydroxyl group under physiological conditions was proposed [37] , [57] . The conserved histidine residue in these two glycosyltransferases probably facilitated the reaction by forming (and thus stabilizing) hydrogen bonds with the deprotonated substrates or glycoside products.…”
Section: Structural Insights Into Plant Ugtsmentioning
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