18O Studies of the 2-Ketoglutarate-dependent Sequential Oxygenation of Thymine to 5-Carboxyuracil

Abstract

Abstract The sequential oxygenation of thymine to 5-hydroxymethyluracil, 5-formyluracil, and 5-carboxyuracil, coupled to the oxidative decarboxylation of 2-ketoglutarate, has been studied in 18O2 experiments with an enzyme preparation from Neurospora crassa strain STA 4 (FGSC 262 A) and with ferrous ion, ascorbate, and catalase as cofactors. Three separate experiments were performed: (1) with thymine and 2-ketoglutarate, (2) with 5-hydroxymethyluracil and 2-ketoglutarate, and (3) with 5-formyluracil and 2-ketoglutarate. The 18O content was assessed by means of mass spectrometry after gas chromatographic separation of the trimethylsilyl derivatives of 5-hydroxymethyluracil (Experiment 1) and 5-carboxyuracil (Experiment 3) as well as of succinic acid (Experiments 1 to 3). The results show the incorporation of one atom of molecular oxygen in the products. It is concluded that the enzyme(s) catalyzing the three reactions belong(s) to the class of oxygenases for which a 2-keto acid functions for oxygen reduction.