From the youngest first leaf pairs of periwinkle leaves.Phylogenetic analyses based on the deduced amino acid sequences of Gr-UGT6, Cr-UGT7, and Cr-UGT8 suggested that while Cr-UGT6 and Cr-UGT8 both belonged to group G, CrUGT7 belonged to group H of Family 1 PSPGs (see Supplemental Figure 1 online). Functionally characterized members of group G are involved in the biosynthesis of the iridoid geniposide (GjUGT2) in gardenia, cyanogenic glucosides (UGT85B1) in sorghum (Sorghum bicolor), cytokinins (UGT85A1) in Arabidopsis thaliana, and in an undisclosed reaction in Lonicera japonica (LjUGT12). Members of group H are involved in the biosynthesis of steviosides (UGTG1) in Stevia rebaudiana, cytokinins (UGTC1) in Arabidopsis, and benzoaxizones (Zea Bx8) in maize (Zea mays). The amino acid sequence comparisons showed 78, 33, and 40 sequence identity of Cr-UGT6, Cr-UGT7, and Cr-UGT8, respectively with the gardenia iridoid-specific glucosyltransferase (Gj-UGT2 or UGT85A24). While these results might suggest that the best candidate for an iridoid-specific GT was UGT6, we decided to functionally characterize all three GTs in order to compare their biochemical properties and their substrate specificities. Functional Characterization of Recombinant UGTs To examine the catalytic function of UGT6-8, their open reading frames were expressed in E.Custom Peptide Synthesis coli as N-terminal fusion proteins with a His6-tag. After purifying each recombinant UGT by nickelnitrilotriacetic acid affinity chromatography, they were assayedFigure 2. Differential Conversions of 7-Deoxyloganetic Acid and 7-Deoxyloganetin by Recombinant UGT6, UGT7, and UGT8 to 7-Deoxyloganic Acid and 7-Deoxyloganin. Iridoid substrates (1 mM) were incubated with each rUGT in the presence of 5 mM UDP-Glc for 2 h at 30 , and the reaction mixture was subjected to HPLC analysis as described in Methods. The arrowheads indicate the respective reaction products (7-deoxyloganic acid or 7-deoxyloganin) being made when recombinant enzymes were incubated with their respective iridoid substrates.The Plant CellTable 1. Kinetic Parameters of rUGT6;8 toward Iridoid Aglycones and UDPG Kinetic Parameters Protein 7-Deoxyloganetic Acid UGT6 UGT7 UGT8 7-Deoxyloganetin UGT6 UGT7 UGT8 UDP-Glc UGT6 UGT7 UGT8 Km (mM) * 0.088 6 0.022 0.202 6 0.030 1.99 6 0.74 0.117 6 0.025 0.120 6 0.013 5.38 6 1.99 kcat (s21) * 0.130 6 0.015 0.0355 6 0.0068 0.00493 6 0.00303 0.0320 6 0.0172 0.00512 6 0.00169 0.325 6 0.051 kcat/Km (M21 s21) * 1523.3 6 222.7 179.9 6 54.5 2.35 6 0.58 291.8 6 103.3 42.0 6 10.4 64.4 6 16.Each data set represents the mean 6 SD from triplicate measurements. The purity of the UGTs used in the kinetic analyses was confirmed by Coomassie blue staining of gels after SDS-PAGE, which made it possible to estimate kcat.Glucose 1-dehydrogenase The alternative substrate concentrations used for UDP-Glc or iridoids were 5 and 0.PMID:24563649 5 mM, respectively, for saturation curves. No activity; *, activity too weak for kinetic assay.for their O-glucosyltransferase activity using 7-deoxyloganetic acid and 7-deoxyloganetin as acceptor substrates in the presence of the UDP-Glc donor (Figure 2). UGT6 rapidly and efficiently converted 7-deoxylaganetin to a product with an identical retention time and UV spectrum as 7-deoxyloganin, whereas no reaction product was detected with 7-deoxyloganetic acid as substrate. UGT8 effectively produced 7-deoxyloganic acid from 7-deoxylaganetic acid, while no such conversion was detected when 7-deoxyloganetin was used as a sugar accept.
