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HPLC analyses show the evolution of product formation in ancestral and modern-day Citrus XMT enzymes. CisAncXMT2 converts xanthine to 1-methylxanthine but not 3-methylxanthine (Insets). In contrast, CisAncXMT2 P25S and CisXMT1 methylate xanthine to both 1-methylxanthine and 3-methylxanthine, which are further converted into theophylline. Enzyme assays were conducted using 2 mM xanthine, and products were measured by absorbance at 272 nm. mAU, milliabsorption units.
