An Ambidextrous Polyphenol Glycosyltransferase PaGT2 from Phytolacca americana

Abstract

The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.

Structure–function relationship of terpenoid glycosyltransferases from plants

“…Currently, 24 crystal structures of plant UGTs family 1 are available these include AtUGT72B1, 53 AtUGT74F2, 54 and AtUGT89C1 55 from Arabidopsis thaliana , CtUGT78K6 from Clitoria ternatea , 56 FeUGT708C1 from Fagopyrum esculentum , 57 GgCGT from Glycyrrhiza glabra , 58 UGT73P12 from Glycyrrhiza uralensis (; 7c2x), LpCGTa and b from Landoltia punctate , 59 MtUGT71G1, 60 MtUGT85H2, 61 and MtUGT78G1 62 from Medicago truncatula , Os79 from Oryza sativa , 63 PtigS from Persicaria tinctoria , 64,65 PaGT2 66 and 3 67 from Phytolacca americana , SbCGTa and b from Scutellaria baicalensis , 59 mogrol glucosyltransferase SgUGT74AC1 and AC2 from Siraitia grosvenorii , 39,68 SrUGT76G1 from Stevia rebaudiana , 69–71 C -glycosyltransferase from Trollius chinensis , 72 VvGT1 from Vitis vinifera , 32 and ZmCGTa from Zea mays 59 (). The 3-dimensional structures show remarkable conservation in their secondary and tertiary structures.…”

Glycosyltransferases: Mining, engineering and applications in biosynthesis of glycosylated plant natural products

“…2 , respectively), while InGTase1 ( Ipomoea nil ) [ 41 ] can catalyze phenylpropanoids and alkaloids ( 7 in Fig. 2 ), PaGT2 ( Phytolacca americana ) [ 42 ] can catalyze phenylpropanoids and polyketides ( 9 in Fig. 2 ), and SaGT4A ( Solanum aculeatissimum ) [ 43 ] can catalyze terpenoids and alkaloids ( 10 in Fig.…”

Structural basis for substrate recognition in the Phytolacca americana glycosyltransferase PaGT3

“…Moreover, the mutation of His20 to Ala or Asp has been shown to completely impair the activity of the enzyme (Ozaki et al, 2012). Another UGT from P. americana, PaGT2, has been shown to possess two catalytic histidines: the conserved catalytic histidine His18 and the alternate catalytic residue His81 (Maharjan, Fukuda, Shimomura et al, 2020). The mutation of either of the catalytic histidines in PaGT2 was compensated by another catalytic histidine, which helped to retain the catalytic activity of the enzyme.…”

“…In UGT78K6 from C. ternatea, Asp367, Asp181 and the mainchain carbonyl O atom of Pro78 stabilize kaempferol, forming hydrogen bonds to the C5, C7 and C4 0 hydroxyl groups of the acceptor molecule, respectively (Hiromoto et al, 2015). Also, in PaGT2 it has been shown that the 3 0 -OH and 4 0 -OH groups of the piceatannol molecule are stabilized by hydrogen bonds to His81 and Glu82, respectively (Maharjan, Fukuda, Shimomura et al, 2020). However, in PaGT3 the acceptor capsaicin is mainly stabilized by hydrophobic interactions and the only possible hydrogen bond is observed to the catalytic His20.…”

“…Most of these UGT structures are in complexes with flavonoid molecules, such as VvGT1 from Vitis vinifera with kaempferol/quercetin (Offen et al, 2006), UGT78G1 from Medicago truncatula with myricetin (Modolo et al, 2009) and UGT78K6 from Clitoria ternatea with delphinidin/petunidin/kaempferol (Hiromoto et al, 2015). Recently, crystal structures of some UGTs that glycosylate other phenolic compounds have also been determined in complex with the corresponding acceptor substrates, such as UGT76G1 from Stevia rebaudiana with rebaudioside A/ rubusoside (Yang et al, 2019), PaGT2 from Phytolacca americana with resveratrol/pterostilbene (Maharjan, Fukuda, Shimomura et al, 2020) and Os79UGT from Oryza sativa with trichothecene (Wetterhorn et al, 2017). Surprisingly, some UGTs can recognize and glycosylate a range of compounds.…”