Fig. 4: Structure-activity relationship studies identify amino acids crucial for GAME8 stereospecificity.
From: Enzymatic twists evolved stereo-divergent alkaloids in the Solanaceae family
a Superposition between SlGAME8 (orange) and SmGAME8 (blue) and molecular docking of 22S-hydroxycholesterol glucuronide in the enzymes active site. Amino acids within 10 Å from the substrate metabolite are visualized, as well as twelve amino acid loop present only in SmGAME8. b GAME8 active site with designated differential amino acid between SmGAME8 and SlGAME8. Red arrow indicates substrate attack from the side of heme molecule. c Multiple sequence alignment of active sites’ amino acid sequences from multiple GAME8 enzymes with 25S (red) or 25R (purple) stereospecificity (for more details see Supplementary Data 1). Amino acid substitutions are indicated with arrows. d Extracted ion chromatograms depicting production of tomatidenol by SlGAME8 WT and solasodine by SmGAME8 and SlGAME8-mut (8 amino acids replaced) in N. benthamiana.
