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HPLC analyses show that CisAncXMT2 P25S converts 1-methylxanthine to theophylline instead of paraxanthine, like its ancestor, CisAncXMT2. Enzyme assays were conducted using 200 µM 1-methylxanthine, and products were measured by absorbance at 272 nm. Unlabeled peaks in Middle and Lower are unidentified molecules found in both the enzyme assay and negative control to which no xanthine alkaloids were added.
