Highly Promiscuous Flavonoid 3-O-Glycosyltransferase from

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Highly Promiscuous Flavonoid 3‑O‑Glycosyltransferase from Scutellaria baicalensis Zilong Wang,†,§ Shuang Wang,†,§ Zheng Xu,‡,§ Mingwei Li,‡ Kuan Chen,† Yaqun Zhang,† Zhimin Hu,† Meng Zhang,† Zhiyong Zhang,*,‡ Xue Qiao,*,† and Min Ye*,† †

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State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China ‡ Hefei National Laboratory for Physical Science at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei 230026, China S Supporting Information *

ABSTRACT: A highly regio-specific and donor-promiscuous 3O-glycosyltransferase, Sb3GT1 (UGT78B4), was discovered from Scutellaria baicalensis. Sb3GT1 could accept five sugar donors (UDP-Glc/-Gal/-GlcNAc/-Xyl/-Ara) to catalyze 3-O-glycosylation of 17 flavonols, and the conversion rates could be >98%. Five new glycosides were obtained by scaled-up enzymatic catalysis. Molecular modeling and site-directed mutagenesis revealed that G15 and P187 were critical catalytic residues for the donor promiscuity. Sb3GT1 could be a promising catalyst to increase structural diversity of flavonoid 3-O-glycosides.

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could be 5−20-times higher for the favored donors than for other donors (Table S1).10 UGT78D2 could accept both UDP-glucose and UDP-N-acetylglucosamine (GlcNAc), but its compatibility with other sugar donors is not known.11 On the other hand, the reported 3GTs only showed high conversion rates with a few substrates. For example, FaGT6 catalyzed 3-hydroxyflavone, galangin, and isorhamnetin with a conversion rate of 80%−100%, while the conversion rates for myricetin, kaempferol, quercetin, and morin were only 10%− 40%. In this work, we report a flavonoid 3-O-glycosyltransferase from Scutellaria baicalensis with high donor- and acceptorpromiscuity. It could regio-specifically accept at least 17 flavonol and anthocyanidin substrates to produce 3-Oglycosides and could accept five different sugar donors (UDP-Glc, -Gal, -GlcNAc, -Xyl, -Ara) at high conversion rates. Scutellaria baicalensis Georgi is a popular medicinal plant worldwide. Its roots are used as the traditional Chinese medicine Huang-Qin. It contains several flavonoid 3-Oglycosides such as kaempferol 3-O-glucoside, indicating the presence of 3GTs.12 Thus, we analyzed the transcriptome data (SRR3123399, SRR3130396, SRR3727161, SRR367956, and SRR3367955) of Huang-Qin to search for 3GTs. Two known GT genes from S. baicalensis (SbUBGAT, EF512580.1 and UGT88D1, AB479151.1) were used as templates in BLASTn search. More than 20 genes with open reading frames (ORF) were obtained. Among them, one candidate gene (Sb3GT1, evalue 5e−4) was tentatively identified as 3-O-glycosyltransferase by NCBI BLAST. The coding region of Sb3GT1 was cloned

lavonoid 3-O-glycosides are popular phenolic compounds in plants. They are widely used in pigments, cosmetics, and natural medicines.1 They show antioxidative, antiinflammatory, and antiviral activities.2 The sugar moiety contributes to structural diversity of flavonoid 3-O-glycosides. They could be glucose (Glc), galactose (Gal), rhamnose (Rha), xylose (Xyl), arabinose (Ara), or apiose (Api) according to previous reports.3 Interestingly, the glycosyl group could remarkably affect the bioactivities. For example, quercetin 3-O-xyloside showed higher inhibitory activities against human dihydroflolate reductase than 3-O-glucoside, 3O-galactoside, and 3-O-rhamnoside.4 Thus, increasing the glycosyl diversity of flavonoid 3-O-glycosides could be an effective way to obtain novel bioactive natural products. Given the multiple hydroxyl groups on most flavonol backbones, regio-specific biocatalytic reactions mediated by glycosyltransferases may be more promising than chemical synthesis.5 Flavonoid 3-O-glycosyltransferases (3GTs) use flavonols, dihydroflavonols, or anthocyanidins as acceptors and mainly use uridine 5′-diphosphate (UDP)-sugar as donors. Plants are the major source of 3GTs. More than 15 plant flavonoid 3GTs have been reported thus far. However, specific and efficient 3GTs with high promiscuity are still lacking. On one hand, the reported 3GTs show strict selectivity toward sugar donors. For example, VvGT1, DiCGT, and FaGT6 could only accept UDP-glucose, but not UDP-galactose;6 F3GalT, DcUCGalT1, and AgUCGalT1 use UDP-Gal, however, but not UDP-Glc;7 EpPF3RT and UGT78D1 utilize only UDP-rhamnose;8 and UGT78D3 only accepts UDP-arabinose.9 Although AcGaT and UF3GaT could accept different sugar donors, they showed obvious preference among the donors. The catalytic ability © XXXX American Chemical Society

Received: February 10, 2019

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DOI: 10.1021/acs.orglett.9b00524 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

exhibited an [M-H]− ion at m/z 447, which is 162 Da greater than that of 1. The [M−H]− ion could produce abundant [Y0−H]−• ion at m/z 284 in the tandem mass spectrometry (MS/MS) spectrum, which was diagnostic for 3-O-glycosides.3 Compound 1a was finally identified as kaempferol 3-Oglucoside by comparing with a reference standard. The reaction conditions were then optimized. Sb3GT1 exhibited its maximum activity at pH 9.0 (50 mM Tris-HCl) and 45 °C. The reaction was independent of divalent metal ions (Figure S2). The apparent Km value for 1 was 24.8 μM (Figure S3). All other reactions were carried out with the optimized conditions (50 mM Tris-HCl with pH 9.0; 0.5 mM sugar donor; 0.1 mM substrate; 4 μg purified Sb3GT1; 45 °C, 1 h). To explore the substrate promiscuity of Sb3GT1, 30 compounds were tested using UDP-Glc as sugar donor (Figure 2, Figure S4). The reaction mixtures were analyzed by liquid chromatography coupled with mass spectrometry (LC−MS) (Figures S5−S20). Sb3GT1 showed high conversion rates (>95%) for flavonols (1−13), flavonol glucosides (14−16), and cyanidin (17). However, flavones (20−22), isoflavones (23, 24), (dihydro)chalcones (25, 26), and other types of compounds (27−30) could not be accepted by Sb3GT1. To further explore the donor promiscuity of Sb3GT1, we tested four other sugar donors, that is, UDP-Xyl, UDP-Gal, UDP-Ara, and UDP-GlcNAc. Surprisingly, Sb3GT1 showed high sugar donor promiscuity when catalyzing the 17 substrates (Figure 2, Figure S5−S20). While all the substrates could accept UDP-Glc efficiently, 1−6, 14, 15, and 17 accepted UDP-Gal at conversion rates of 70%−98%. All the substrates could efficiently utilize UDP-GlcNAc (conversion rates 80%−98%) except for 12 and 13. The conversion rates for 1−6 and 14−15 were >98% when UDP-Xyl was used as the donor. Moreover, compounds 1, 2, 5, 6, and 15 could accept UDP-Ara at conversion rates of 80%−98%.

into pET-28a(+) vector, and the protein was expressed in an E. coli BL21(DE3) strain. Sb3GT1 contains a 1374-bp ORF, which encodes a 457-amino acid polypeptide. It was named as UGT78B4 by the UGT Nomenclature Committee. The enzyme was purified by Ni-NTA affinity chromatography and was analyzed by SDS-PAGE (Figure S1). The function of Sb3GT1 was characterized using kaempferol (1) as the substrate and UDP-Glc as the sugar donor. Highperformance liquid chromatography (HPLC) analysis showed one single product (1a) in the reaction mixture (Figure 1). It

Figure 1. Functional characterization of Sb3GT1. (A) Glycosylation reactions of kaempferol (1) catalyzed by Sb3GT1 using five different sugar donors. (B) HPLC chromatograms of the enzymatic reaction mixtures and the substrate. (C) LC−MS/MS analysis of glycosylated products (1a, 1c, and 1d) in the negative ion mode.

Figure 2. Substrate and donor promiscuity of Sb3GT1. (A) Structures of 1−17 and their 3-O-glycosylated products. (B) Conversion rates of compounds 1−17 with different sugar donors catalyzed by Sb3GT1. ∗ indicates new compounds, and Δ indicates products identified by comparing with reference standards (1b, 2c, and 3b were purified from scaled-up enzymatic reactions in this work). B

DOI: 10.1021/acs.orglett.9b00524 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 3. Molecular modeling and site-directed mutagenesis revealed the critical residues of Sb3GT1. (A) Model of the Sb3GT1 donor binding pocket, showing T16 and T281 stabilizing the sugar donor. UDP-Glc and kaempferol (1) are shown in green. (B) Model of the substrate binding pocket, showing key residues G15, S139, A143, and P187. The model of Sb3GT1 (cyan) and 1 (slate) was aligned with the crystal structure of VvGT1 (orange) and 1 (magenta). (C) Catalytic capabilities of Sb3GT1 mutants to accept different UDP-sugar donors.

UDP-Glc, Zone B was near the diphosphate group of UDPGlc, and Zone C included the residues around the uridine tail of UDP-Glc. In Zone B, two residues were critical to stabilize UDP-Glc in the binding pocket, namely T16 on loop 1 and T281 on loop 2 (Figure 3A). Both residues are conserved in the crystal structures of UGT78G1 and VvGT1 (Figure S61). Accordingly, we mutated these residues in Sb3GT1 (T16A/ T281A mutant) and detected a complete loss of function toward all donors (Figure 3C). In Zone C, the putative secondary plant glycosyltransferase (PSPG) motif, which contains W333-Q336 in Sb3GT1, is conserved with UGT78G1 and VvGT1 (Figure S62). These results indicated reliability of the protein model. The structure of Sb3GT1 showed high similarity to the crystal structures of UGT78G1 and VvGT1, as shown by the overall structural alignments (Figures S63, S64). UGT78G1 is a multifunctional triterpene/flavonoid GT, while VvGT1 is a specific 3GT preferring UDP-Glc donor. Thus, we examined the difference between Sb3GT1 and VvGT1. By checking the residues around the binding pocket (Zone A), 18 residues were found within a 5-Å distance to kaempferol (Figure S65). Among them, four residues were different from VvGT1: G15/ S18, S139/A142, A143/S146, and P187/Q188 (Sb3GT1/ VvGT1, Figure 3B). They might be critical for the sugar donor promiscuity of Sb3GT1. The S18 residue in VvGT1 could interact with kaempferol C4−O via a hydrogen bond (2.7 Å), which may stabilize the substrate for reaction.16 When S18 was replaced by G15 in Sb3GT1, a broader space between the substrate and the UDPsugar was formed, and it may allow the binding of structurally different sugar donors such as UDP-Gal. Similarly, A143 and P187 residues in Sb3GT1 have smaller side chains than S146 and Q188 in VvGT1, respectively. This may allow higher flexibility and variability for the substrates. The surface view of VvGT1 and Sb3GT1 around the substrate binding pocket reveals a broader interspace in Sb3GT1 (Figure S66). Furthermore, we calculated the pocket volume near the phydroxyphenyl tail of kaempferol.17 The result showed the pocket volume of Sb3GT1 was approximately 20% bigger than that of VvGT1 (Figure S67). We then performed MD simulations for different donors.18 As a result, both UDP-Glc and UDP-Gal could bind with Sb3GT1, and the stabilities were similar (RMSD < 2.5 Å vs RMSD < 3.0 Å, Figure S68), which was consistent with our experimental results. To prove our hypothesis, a series of Sb3GT1 mutants was constructed, including G15S, G15F, G15 V, S139A/A143S,

For all the above catalytic reactions, Sb3GT1 produced one single product. A total of 19 products were unambiguously identified as 3-mono-O-glycosides, either by comparing with reference standards, or by HR-MS and NMR spectroscopic analyses following purification from scaled-up enzymatic reactions (1.2−3.8 mg substrates were used) (Figures S21− S58). For example, 1c was identified as kaempferol 3-OGlcNAc according to the HMBC correlation between C-3 (δC 133.4) and H-1′′ (δH 5.6).11 Five products (1c, 3c, 4a, 4b, 4c) are new compounds. The other products were tentatively characterized as 3-O-glycosides by MS/MS according to the diagnostic [Y0−H]−• fragment ions in MS/MS spectra (Figure 1).3 The above data demonstrated that Sb3GT1 could efficiently and regio-specifically catalyze the glycosylation of flavonols and cyanidin at 3-OH. To the best of our knowledge, Sb3GT1 has remarkably broader donor-promiscuity and substrate-promiscuity than previously reported flavonoid 3-O-glycosyltransferases.6,13 To explain the promiscuity of Sb3GT1, molecular modeling and site-directed mutagenesis were conducted. The protein model for Sb3GT1 was established by homologous molecular modeling using SWISS-MODEL.14 The crystal structure of UGT78G1 (PDBID: 3HBJ), which exhibits the highest reliability with a GMQE (global model quality estimation) value of 0.78, was used as the modeling template.15 It shares amino acid sequence identity of 48% with Sb3GT1. However, the UGT78G1 template only carries UDP ligand in the crystal structure. To better understand the binding mode of Sb3GT1 with kaempferol, we found another homologue VvGT1 and its crystal structure with UDP-2-deoxy-2-fluoro glucose and kaempferol (PDBID: 2C1Z).16 This protein shares amino acid sequence identity with Sb3GT1 of 50% and showed a GMQE of 0.76. With root−mean−square deviation (RMSD) of 98% (Figure S69). In summary, Sb3GT1 is an efficient and regio-specific glycosyltransferase to catalyze 3-O-glycosylation of flavonols and cyanidin. It showed unprecedented donor- and substratepromiscuity to accept five sugar donors (UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-Xyl, and UDP-Ara) and at least 17 substrates. The conversion rates were >98% when kaempferol, quercetin, patuletin, and kaempferol 7-O-glucoside were used as the substrates. The promiscuity of Sb3GT1 may result from G15 and P187 residues, which change the sugar donor binding pocket when binding with the acceptors. Sb3GT1 could be a promising catalyst to increase structural diversity of flavonoid 3-O-glycosides through either enzymatic reactions or whole cell biotransformations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00524. Molecular cloning, expression, purification of Sb3GT1; effects of reaction time, pH, temperature, divalent metal ions; determination of kinetic parameters; enzyme assays of Sb3GT1; scaled-up reactions; 1H and 13C NMR data of glycosylated products; computational modeling and molecular docking; site-directed mutagenesis (PDF) Accession Codes

GenBank MK577650 (Sb3GT1, UGT78B4).

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhiyong Zhang: 0000-0001-8202-9672 Xue Qiao: 0000-0002-8771-7877 Min Ye: 0000-0002-9952-2380 Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Nos. 81725023, 81891010/81891011, D

DOI: 10.1021/acs.orglett.9b00524 Org. Lett. XXXX, XXX, XXX−XXX

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