Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential antifungal targets

Test of ara-2″-fluoro NAD⁺ as substrate for fungal Tpt1 enzymes. Reaction mixtures (10 µL) containing 100 mM Tris-HCl, pH 7.5, 2 mM DTT, 0.2 µM (2 pmol) 5′ ³²P-labeled 6-mer 2-PO₄ RNA, 50 µM NAD⁺ or ara-2″F-NAD⁺ (shown in panel C) where indicated by +, and either 10 fmol Tpt1 (A) or 5 pmol Tpt1 (B) were incubated at 37°C for 30 min. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The species corresponding to the 2′-phosphate substrate, 2′-OH product, and 2′-phospho-ADParaF intermediate are indicated on the left.