Figure 8.

Determinants of Sugar Donor Sugar Specificity of AsAAT1.

(A) Model of AsAAT1 with bound UDP-Ara (carbons of the Ara numbered). The protein is represented in green ribbons with the PSPG motif in salmon, including the side chains of highly conserved residues. The His⁴⁰⁴ and Pro¹⁵⁴ residues are shown in magenta. Potential hydrogen bonds are shown with yellow dots, and the distance between P154 and C-5 of UDP-Ara with gray dots. The homology model was generated using I-TASSER software (Yang et al., 2015), based on the crystal structure of M. truncatula UGT71G1 complexed with UDP-Glc (PDB: 2ACW). The loop shown in orange was reconstructed using MODELER (Sali and Blundell, 1993). UDP-Ara was inserted into the active site and the complex was relaxed by energy minimization using GROMACS.

(B) Comparison of the glycosylation activity of the AsAAT1 wild-type and mutant enzymes when supplied with each of the four sugar donors (UDP-Ara, UDP-Gal, UDP-Xyl, or UDP-Glc). Initial velocities were measured using 30 µM deglycosylated avenacin A-1 as acceptor and 5 µM UDP-sugar donor using five time points. The heights of the bars are drawn relative to the highest activity observed for each recombinant enzyme (mean ± sd, n = 3). Activities reported above each bar are in nM.min⁻¹.

(C) HPLC-CAD analysis of extracts from N. benthamiana leaves expressing SAD1 and SAD2 together with GFP (black), wild-type AsAAT1 enzyme (blue), AsAAT1-H404Q (green), AsAAT1-P154S (red), and AsAAT1-H404Q-P154S (orange). The top trace (in gray) shows the products of in vitro reaction of EpHβA with the four sugar donors (reactions performed separately and then pooled) for reference. LC-MS analysis was used to confirm the identities of the new products (Supplemental Figure 8C). The IS is digitoxin (1 mg/g dry weight).