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

Fungal Tpt1s efficiently remove an internal 2′-phosphate from a DNA substrate. Reaction mixtures (10 µL) containing 100 mM Tris-HCl, pH 7.5, 2 mM DTT, 1 mM NAD⁺, 0.2 µM (2 pmol) 5′ ³²P-labeled 6-mer DNA with a single internal ribonucleotide 2-PO₄ (shown in A with deoxynucleotides in italics), and CauTpt1 (A), CalTpt1 (B), AfuTpt1 (C), or CimTpt1 (D) as specified on the x-axes were incubated at 37°C for 30 min. The extents of formation of the 2′-OH product are plotted as a function of input Tpt1. Each datum is the average of three independent titration experiments ±SEM.