Fig. 2: GAME8 is a source of SGA stereodiversity in Solanum species.
From: Enzymatic twists evolved stereo-divergent alkaloids in the Solanaceae family
a Phylogenetic analysis of GAME8 coding sequences (CDSs) from multiple Solanaceae species. CYP72B1 (brassinosteroid C26 hydroxylase) from tomato was used as an outgroup, and GAME6 CDSs (cholesterol-GlcA C22 hydroxylase) were used to stabilize the tree. Two clades with distinct stereo specificities are marked with either red or purple braces. Red and purple disks designate C25 stereo specificity of in planta tested (i.e., in N. benthamiana) GAME8s; yellow triangles label duplicated proteins with divergent activities clustering to different clades, while blue and green triangles label proteins with opposite activities clustering in one clade. Pale purple disk designates GAME8 enzyme lacking C26/C27 hydroxylase activity in planta. b Proposed SGA aglycones biosynthetic pathway with C25 chirality marked either in red (25S) or purple (25R). c Extracted Ion Chromatograms depicting solasodine (25S) and tomatidenol (25R) production by selected Solanum GAME8 enzymes in N. benthamiana. d Simplified chemical structures of 22S,26(27)-dihydroxycholesterol-d7 glucuronides with C25 configuration assigned. e Extracted ion chromatograms depicting accumulation of isotopically labeled 22S,26(27)-dihydroxycholesterol-d7 glucuronides (25S or 25R) confirming GAME8 activity as C26 or C27 hydroxylase. Retention time difference between deuterium-labeled and non-labeled metabolites is due to the deuterium isotope effect.
