Fig. 5. Origins of OsUGT91C1 promiscuity.

a OsUGT91C1 binds Reb E with the R2 end at the active site is ready to add glucose 2-1-R2 to glucose 1-R2. The protein is represented in the surface, and the loose-fitting substrate tunnel of OsUGT91C1 is delimited by white dashed lines. The catalytic residue His27 is colored yellow (marked with a red asterisk), and the residues Phe208 and Glu283 are colored salmon on the surface. Phe379 cannot be seen as a result of the cutaway surface presentation, and this residue is shown in Fig. 3c–e. The positions of the R1 and R2 ends, glucose units, the reactive 2-hydroxyl, and 6-hydroxyl are labeled with a circle shadow for clarity. b OsUGT91C1 is able to bind the aglycone in the opposite orientation (the transition is shown in the red dashed box) so that the R1 end is now at the active site positioned for the addition of glucose 2-1-R1 to glucose 1-R1. The figure uses the same design as a. c After the addition of glucose 2-1-R1, glucose 1-R1 has flipped by 180° from that seen in b (the transition is shown in the red dashed box). As a result, glucose 2-1-R1 is held in a new pocket, freeing up the enzyme to bind fresh UDP-glucose and positioning the 6-OH of glucose 1-R1 for catalysis. The figure uses the same design as a. d Structurally related β (1–3) glycosyltransferase UGT76G1 (Fig. 1b) binds the aglycone in a different location. As a result, 3-OH, not 2-OH, is positioned for catalysis. The figure follows the design of a with color differences. The orientation of UGT76G1 here matches that of OsUGT91C1 in a–c. In UGT76G1, His25 serves as the catalytic base and is colored yellow (marked with a red asterisk) on the surface.