Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales Journal Article
| Authors: | Ghosh, S.; Wimberly-Gard, G.; Jacewicz, A.; Schwer, B.; Shuman, S. |
| Article Title: | Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales |
| Abstract: | Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2′′,3′′-cyclic-PO4 and 5′′-OH ends of tRNA exons into the 3′′-OH,2′′-PO4 and 5′′-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3′′-OH,2′′-PO4 and 5′′-PO4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5′′-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg2+ that illuminates the basis for guanosine nucleotide specificity. © 2024 Ghosh et al. |
| Keywords: | controlled study; genetics; nonhuman; protein domain; metabolism; in vivo study; in vitro study; enzymology; enzyme activity; recombinant enzyme; rna; chemistry; dna; enzyme phosphorylation; amino acid sequence; biological activity; enzyme analysis; saccharomyces cerevisiae; guanosine; sequence alignment; crystal structure; models, molecular; crystallography, x-ray; transfer rna; rna, transfer; x ray crystallography; enzyme structure; guanosine triphosphate; phosphate; rna end healing; trna splicing; polynucleotide 5' hydroxyl kinase; polynucleotide 5'-hydroxyl-kinase; fungal protein; molecular model; rna splicing; nucleotide; genetic complementation; divalent cation; species; magnesium; fungal proteins; rhizopus; article; fungal pathogen; mucorales; lichtheimia corymbifera; mucor circinelloides |
| Journal Title: | RNA |
| Volume: | 30 |
| Issue: | 12 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2024-12-01 |
| Start Page: | 1674 |
| End Page: | 1685 |
| Language: | English |
| DOI: | 10.1261/rna.080247.124 |
| PUBMED: | 39357987 |
| PROVIDER: | scopus |
| PMCID: | PMC11571804 |
| DOI/URL: | |
| Notes: | The MSK Cancer Center Support Grant (P30 CA008748) is acknowledged in the PubMed record and PDF. Corresponding MSK author is Stewart Shuman -- Source: Scopus |
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