Functional characterization of three flavonoid glycosyltransferases from Andrographis paniculata

Andrographis paniculata is an important traditional medicinal herb in South and Southeast Asian countries with diverse pharmacological activities that contains various flavonoids and flavonoid glycosides. Glycosylation can transform aglycones into more stable, biologically active and structurally diverse glycosides. Here, we report three glycosyltransferases from the leaves of A. paniculata (ApUFGTs) that presented wide substrate spectra for flavonoid glycosylation and exhibited multi-site glycosylation on the substrate molecules. They acted on the 7-OH position of the A ring and were able to glycosylate several other different types of compounds. The biochemical properties and phylogenetic analysis of these glycosyltransferases were also investigated. This study provides a basis for further research on the cloning of genes involved in glycosylation from A. paniculata and offers opportunities for enhancing flavonoid glycoside production in heterologous hosts. These enzymes are expected to become effective tools for drug discovery and for the biosynthesis of derivatives via flavonoid glycosylation.

YL, 0000-0001-9942-0803; WG, 0000-0003-3081-9642 Andrographis paniculata is an important traditional medicinal herb in South and Southeast Asian countries with diverse pharmacological activities that contains various flavonoids and flavonoid glycosides. Glycosylation can transform aglycones into more stable, biologically active and structurally diverse glycosides. Here, we report three glycosyltransferases from the leaves of A. paniculata (ApUFGTs) that presented wide substrate spectra for flavonoid glycosylation and exhibited multi-site glycosylation on the substrate molecules. They acted on the 7-OH position of the A ring and were able to glycosylate several other different types of compounds. The biochemical properties and phylogenetic analysis of these glycosyltransferases were also investigated. This study provides a basis for further research on the cloning of genes involved in glycosylation from A. paniculata and offers opportunities for enhancing flavonoid glycoside production in heterologous hosts. These enzymes are expected to become effective tools for drug discovery and for the biosynthesis of derivatives via flavonoid glycosylation.

cDNA synthesis and gene cloning
UGTs were screened from A. paniculata transcriptome databases. To clone permissive ApUFGTs from A. paniculata, leaves of A. paniculata were treated with MeJA for 48 h prior to RNA isolation. The extracted RNA (Thermo Fisher Scientific, CA, USA) was used to synthesize cDNA using a PrimerScript TM RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's protocol. Fulllength coding sequences of the selected UGTs were amplified by PCR using specific primers designed by Primer Premier 5.0 software (table 1). PCR was performed in a 100 ml scale using KOD-Plus-Neo (TOYOBO, Japan) at 948C for 2 min; 35 cycles of 988C for 10 s, annealing at 558C for 30 s and extension at 688C for 1 min; a final extension at 688C for 5 min. PCR products were purified using a GeneJET Gel Extraction Kit (Thermo Scientific, USA) and ligated into the N-terminal MBP fusion expression vector HIS-MBP-pET28a ( provided by Dr Xiaohong Zhang; HIS, histidine; MBP, maltose-binding protein) that had previously been digested with the restriction enzymes BamHI and SalI according to the protocol accompanying the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech). The conjugates were transformed into Trans1 T1 phage-resistant chemically competent cells (TransGen Biotech, Beijing, China). The recombinant plasmids were obtained by screening positive clones and sequencing.

Sequence alignment and phylogenetic analysis
DNAMAN software was used to carry out the multiple alignment. ClustalW analysis software was used to compare the amino acid sequences of the flavonoid glycosyltransferases from other plant sources, and a phylogenetic tree was constructed using MEGA 7.0 software. Branch support was evaluated using bootstrap analysis with 1000 replicates [51].

Heterologous expression and affinity purification of ApUFGTs
The recombinant plasmids were transformed into Escherichia coli Transetta (DE3) expressing competent cells (TransGen Biotech, Beijing, China). The monoclonal colonies were identified and transferred to Luria-Bertani (LB) medium containing kanamycin (50 mg ml 21 ). When the density of the host bacteria (OD 600 ) reached 0.6-1.0 following incubation at 378C, an appropriate IPTG inducer (final concentration of approx. 1 mM) was added to induce culturing at a low temperature (168C) for 12 h. The samples were subjected to centrifugation at 48C for 20 min, suspension in lysis buffer (50 mM PBS (pH 7.4), 1 mM EDTA, 10% glycerol and 1 mM PMSF), disrupted by sonication in an ice bath (ultrasonic power 5 s, interval 5 s, continuous for 10 min) and followed by centrifugation at 10 000g for 10 min. The crude proteins were filtered through a 0.45 mm membrane, transferred to an Ni-NTA agarose affinity column (Qiagen, WI, USA) and rotated at 48C for 2 h to allow the Ni-NTA to fully bind to the protein. The samples were eluted with different concentrations of imidazole/PB buffer (0.02 M Na 2 HPO 4 -NaH 2 PO 4 (pH 7.4) and 0.5 M NaCl with imidazole concentrations of 50, 100, 200, 300 and 500 mM). The proteins were then concentrated by Amicon Ultra-30 K filters (Millipore, USA), and finally, the buffer was changed to desalting buffer (50 mM Tris-HCl, pH 7.4). The protein concentrations were determined using a modified Bradford protein assay kit (Sangon Biotech, Shanghai, China), and the purified proteins were validated by SDS-PAGE.

Enzyme assays
The reaction system for the ApUFGT activity assay was as follows: a total volume of 100 ml containing 50 mM Tris -HCl ( pH ¼ 8.0), 8 mg purified proteins, 320 mM aglycone and 3200 mM UDP-glucose. The reaction was conducted at 408C for 6 h and twice the volume of methanol was added to terminate the reaction; the mixture was shaken well, centrifuged at 12 000g for 10 min, and then the supernatant was filtered through a 0.22 mm filter and subsequently analysed. The chromatographic analyses were conducted using a Waters Acquity UPLC-I-Class system (Waters Corp., Milford, MA, USA) with an Acquity UPLC BEH C18 column (1.7 mm, 2.1 Â 50 mm). Gradient programmes were used to analyse the reaction mixtures (

Effects of temperature and pH on enzyme activities
The assays of the biochemical properties of temperature and pH were performed by changing each of the reaction conditions. To determine the optimal reaction temperature, the reaction mixtures were incubated at different temperatures (20, 30, 40, 50 and 608C). To study the optimal pH, the enzymatic reactions were performed in various reaction buffers with pH values in the range of 4.0-11.0 ( pH 4.0-7.0, citric acidsodium citrate buffer; pH 7.0-9.0 Tris -HCl buffer; and pH 9.0-11.0, Na 2 CO 3 -NaHCO 3 buffer). All experiments were performed with UDPG as the donor and wogonin (10) as the acceptor in a total volume of 100 ml as described above. All experiments were carried out in triplicate. The mixtures were analysed by UPLC analysis as described in table 2. The total conversion rate was calculated to be 1% of the total peak area of the substrate and product.

Study on the catalytic promiscuity of ApUFGTs
Several drug-like compounds with different types of structures were selected as substrates, including flavones (1-6), a flavonol (7), a flavanone (8), isoflavones (9 and 10), a dihydrochalcone (11), a coumarin (14) and other small molecular aromatic compounds with -OH and -NH 2 groups (12 and 13) (figure 4b).  The scopes of the substrates tolerated by the three recombinant ApUFGTs were systematically studied. For the same substrate, the types and conversion rates of the glycosylation products catalysed by the three glycosyltransferases are different (figure 4a), illustrating the diversity of plant secondary metabolic glycosyltransferases in A. paniculata. For the flavonoids in A. paniculata (1, 2 and 3), the three ApUFGTs all exhibit glycosylation activity and can glycosylate multiple types of hydroxyl groups on certain substrates (1 and 3); there were at least two products in the glycosylation reaction of the two substrates. For certain flavonoids (2, 4, 5 and 6), the three ApUFGTs all exhibit strong positional selectivity, resulting in only one glycosylation product. Other flavonoids can be converted into different multi-site glycosylated products (7)(8)(9)(10)(11). Interestingly, for a non-natural (synthetic) substrate (13), ApUFGT1 and ApUFGT2 can catalyse the formation of N-glycoside bonds, highlighting the potential of these enzymes as multifunctional glycosylation tools.

Biochemical properties of ApUFGTs
Temperature and pH are two of the most important factors affecting enzymatic activity. The effects of different reaction temperatures (20-608C) on the glycosylation of wogonin catalysed by ApUFGTs were investigated. The results showed that the three ApUFGTs all exhibited the highest enzymatic activity at 408C. When the reaction temperature exceeded 408C, the enzymatic activity decreased rapidly with increasing temperature, while the enzymatic activity remained low when the reaction temperature was below 308C ( figure 5). Therefore, the optimum temperatures of the ApUFGTs were all approximately 408C. In the range of pH 7-8, the ApUFGTs all had higher enzymatic activities, but royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 190150 when the pH of the reaction system was below 6 or above 8, the activity decreased significantly ( figure 6). Therefore, the optimum pH values for ApUFGTs were all approximately 8.0.

Discussion
Glycosyltransferases bear considerable importance owing to the fact that glycan moiety forms a necessary element of the plant secondary metabolisms, and can alleviate these disadvantages of chemical glycosylation [30]. With the progress in next-generation sequencing technologies and the reduction in the cost of sequencing, transcriptome analyses have become an important method for identifying the genes that participate in the biosynthesis of natural products, which can provide genetic information, gene expression levels and the basis for subsequent screens of candidate genes [52]. To identify the enzyme responsible for the glycosylation of flavonoids in A. paniculata, timecoursed transcriptome sequencing with MeJA treatment was performed, which may be a powerful tool for the further characterization of UGTs or other genes involved in secondary metabolisms.
Although significant progress has been made recently in the identification of putative UGT genes of many plant species, reports that documented the characterization of UGT family proteins with catalytic promiscuity remain relatively small. Here, we report a total of three UGTs from the leaves of A. paniculata, which exhibited broad substrate tolerance towards multiple flavonoids and could glycosylate various hydroxyl sites on flavonoids in vitro to preferentially form 7-O-glucoside products. The phylogenetic and sequence analysis of ApUFGTs reflect the diversity of glycosyltransferases in the same plant. With the continuous mining of glycosyltransferases in A. paniculata, novel enzymes with glycosylation activity will be identified, and the biosynthetic pathway of active components in A. paniculata will also be elucidated.

Conclusion
Using transcriptome sequencing, we identified and characterized three glycosyltransferases from A. paniculata (ApUFGTs), all of which could glycosylate flavonoids with various structures and preferentially glycosylate the 7-OH of their A ring. These enzymes also exhibited catalytic promiscuity in the glycosylation of different hydroxyl groups on flavonoids. In addition, ApUGT1 and ApUGT2 were capable of catalysing O-, and N-glycosidic bond formation. Biochemical properties and phylogenetic analysis of ApUFGTs were also investigated. These three glycosyltransferases could be effective enzymatic tools for the synthesis of flavonoid glycosides with different types of structures. This study not only holds considerable promise for the resource development of A. paniculata, but also facilitates further enzyme engineering in drug design and the discovery of new active leading compounds.
Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material.