Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

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 <i>Mucor circinelloides</i> 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 <i>Rhizopus azygosporus</i> (RazKIN) and <i>Lichtheimia corymbifera</i> (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.