Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: Essential tRNA splicing enzymes and potential antifungal targets Journal Article
| Authors: | Dantuluri, S.; Schwer, B.; Abdullahu, L.; Damha, M. J.; Shuman, S. |
| Article Title: | Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: Essential tRNA splicing enzymes and potential antifungal targets |
| Abstract: | The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2-PO4 attacks NAD+ to form an RNA-2-phospho-(ADPribose) 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 mutation and in vitro in NAD+-dependent conversion of a 2-PO4 to a 2-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu ofNAD+, 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-PO4 nucleotide with arabinose-2-PO4 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-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity. © 2021 Cold Spring Harbor Laboratory Press. All rights reserved. |
| Keywords: | trna splicing; nicotinamide adenine dinucleotide; antifungal target; 2-phosphotransferase |
| Journal Title: | RNA |
| Volume: | 27 |
| Issue: | 5 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2021-05-01 |
| Start Page: | 616 |
| End Page: | 627 |
| Language: | English |
| DOI: | 10.1261/rna.078660.120 |
| PUBMED: | 33509912 |
| PROVIDER: | scopus |
| PMCID: | PMC8051265 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 June 2021 -- Source: Scopus |
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