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The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2′-PO 4  generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2′-PO 4  attacks NAD +  to form an RNA-2′-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential antifungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens:  Coccidioides immitis , the agent of Valley Fever;  Aspergillus fumigatus  and  Candida albicans , which cause invasive, often fatal, infections in immunocompromised hosts; and  Candida auris , an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal  Saccharomyces cerevisiae tpt1 Δ mutation and in vitro in NAD + -dependent conversion of a 2′-PO 4  to a 2′-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD + , albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2′-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2′-PO 4  nucleotide with arabinose-2′-PO 4  diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2′-phospho-ADP-ribosylated intermediate. Our results implicate the 2′-PO 4  ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.

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