Substrate preferences of ancestral CS-type enzymes reveal the origins of modern-day enzyme activities. (A) Estimated CS gene tree (lnL = −19,220.31407) shows general relationships among sequences from divergent orders of angiosperms. Node labels are shown for reconstructed ancestral enzymes. (B) Xanthine alkaloid substrates tested with each CS enzyme are color-coded to represent structures shown in figure 1. X = xanthine, XR = xanthosine, 1X = 1-methylxanthine, 3X = 3-methylxanthine, 7X = 7-methylxanthine, TP = theophylline, TB = theobromine, PX = paraxanthine, CF = caffeine. (C) Ancestral Paullinia CS enzymes preferred to methylate 7X as shown by the pie charts at nodes M and N but ultimately evolved modern-day enzymes to sequentially methylate X and 3X and catalyze a complete pathway to TB. (D) Ancestral Theobroma CS enzyme at Node O preferred to methylate 7X. After duplication, preference for X evolved in the ancestral enzyme at Node P. From this enzyme, modern-day Theobroma CS enzymes evolved substrate preferences allowing for TB biosynthesis via sequential methylation of X and 3X. (E) Ancestral Camellia CS enzyme of Node Q also preferred to methylate 7X but later evolved divergent modern-day activities shown for C. sinensis TCS1 and TCS2. Inset boxes showing CF pathway network is shaded for ancestral enzymes. The pie charts of figure 2C–E represent mean relative activity with each substrate for the combined ancestral alleles at each node. Enzymes marked with “*” are taken from published studies from other laboratories.