Udp glc

The purified His-tagged recombinant MrSGT was prepared as previously described in our recent publication (Hoang et al. 2016 (link)). The authentic sterols such as β-sitosterol, campesterol and cholesterol (Fig. 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MrSGT (2 μM) was incubated with each of the above three sterols (glycosyl acceptors) and UDP-Glc (Sigma-Aldrich) as a glycosyl donor, in 100 μL reaction buffer (50 mM Tris–HCl, pH 7.4; 5 mM MgCl₂) at 30 °C for 15 min. For the determination of the kinetic parameters for each sterol, the concentration of UDP-Glc was fixed at 0.8 mM, while the concentration of the sterol was varied from 0.05 to 0.6 mM. To measure the kinetic properties with respect to UDP-Glc, the concentration of the sterol was kept constant at 0.6 mM, while the concentration of UDP-Glc was varied from 0.1 to 0.8 mM. All reactions were quenched by the addition of 100 μL of ethyl acetate. After the evaporation of the solvent layer to dryness, aliquots of extracts, reconstituted in methanol, were subjected to HPLC–MS/MS analyses as described below. Each experiment was performed in triplicate, and the reaction mixture containing boiled MrSGT served as control.Fig. 1

Chemical structures of sterols used as acceptors for in vitro MrSGT enzyme reactions

A total of 41 compounds, including flavonoids (1–7), anthraquinones (8), steroids (9–25), terpenoids (26–28), phenolic acids (29–33) and alkaloids (34–41), were used as the sugar acceptors for OsUGT1-catalyzed glycosylation reactions (Figure 2, Table S1). Four UDP-activated nucleotides, namely UDP-Glc, UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-D-xylose (UDP-Xyl) and UDP-l-arabinose (UDP-Ara), were used as the sugar donors. UDP-Glc and UDP-GlcNAc were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). UDP-Xyl and UDP-Ara were enzymatically synthesized as described previously [14 (link),15 (link)]. Two aryl-substituted glycosides, o-Nitrophenyl-β-d-glucopyranoside (oNP-β-Glc) and o-Nitrophenyl-β-d-galactopyranoside (oNP-β-Gal), were purchased from J&K Chemical Ltd (Shanghai, China) and applied as the substrates for transglucosylation or hydrolysis reaction [16 (link)]. The other chemicals were either reagents or analytic grade when available.

Glc-1P, Gal-1P, GlcN-1P, ATP, ITP, UTP, CTP, GTP, ADP-Glc, UDP-Glc, GDP-Glc, inorganic pyrophosphate (PPi) and Fru-1,6-bisP were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest quality available.

M, Q, K, M3Rha, Q3Rha, Q3Gal, K3Gal, K3Rha, flavanols (catechin and epicatechin), flavanones (naringenin and hesperetin), isoflavones (genistein and daidzein), and flavones (apigenin and luteolin) were all HPLC standard and obtained from Aladdin (Shanghai, China). UDP-Rha was bought from Yuanye (Shanghai, China). HPLC-grade acetonitrile and methanol, UDP-Glc, and UDP-Gal were bought from Sigma–Aldrich (St Louis, MO, USA).

The CatD of ENB1 or ENB1^G780R (enb1), as described previously with minor modifications (Olek et al., 2014) (link), was used for the UDP-Glc (Sigma-Aldrich, St. Louis, MO, USA; cat. no. U4625) binding assay. ENB1 or enb1 CatD corresponding to the 1,015–2,523-bp region of ENB1 or enb1 ORF was cloned into the pCold-TF DNA Vector (Takara, Shiga, Japan) that contains an N-terminal His tag and a soluble tag of trigger factor (TF) chaperone. The recombinant His-TF-ENB1 CatD or His-TF-ENB1^G780R CatD was expressed in E. coli Rosetta (DE3) cells by adding 0.5 mM of IPTG when OD₆₀₀ reached 0.8. Recombinant proteins were purified with the BeaverBeads IDA-Nickel (Beaver, Suzhou, China; cat. no. 70501-5) and then labeled with the Monolith Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper, Munich, Germany; cat. no. MO-L011). The microscale thermophoresis assays were conducted using a Monolith NT.115 (NanoTemper, Munich, Germany) machine. The serial concentrations of UDP-Glc were titrated against 100 nM of His-TF-ENB1 CatD or His-TF-ENB1^G780R CatD protein in the optimized buffer (50 mM Tris–HCl, pH 7.4, 150-mM NaCl, 10-mM MgCl₂, 0.05% Tween-20). Each protein was labeled 3 times for three independent tests. All data were analyzed using the MO. Affinity Analysis version 2.3 software. Relevant primer sequences are given in Supplemental Data Set 6.