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-(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 tpt1Δ 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 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′-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.

Molecular architecture of the human tRNA ligase complex

“…RtcB ligases are present throughout all domains of life with notable absences in plants and some fungi, including the model organisms S. cerevisiae and Schizosaccharomyces pombe ( Dantuluri et al, 2021 ; Popow et al, 2012 ). Although their catalytic mechanism is conserved, their substrates and consequently their cellular functions vary considerably ( Englert et al, 2011 ; Popow et al, 2011 ; Popow et al, 2012 ).…”

Structural basis for Tpt1-catalyzed 2′-PO4transfer from RNA and NADP(H) to NAD+

“…1 E ). The Tpt1·ADP interface underscores the basis for NAD + substrate recognition, whereby i) adenosine adopts an anti conformation and the adenosine ribose a C2′ endo pucker; ii) a hydrogen bond from the Met112 amide nitrogen to adenine-N7 imparts purine specificity; iii) nucleobase selectivity for adenine versus hypoxanthine ( 11 ) is conferred by a hydrogen bond from adenine-N6 to the Gly110 carbonyl; iv) the adenosine ribose 2′-OH makes hydrogen bonds to His96-Nε and Thr98-Oγ of the conserved His-Gly-Thr motif; and v) the nonbridging α and β phosphate oxygens are coordinated by the Arg116, Arg134, and His135 side chains ( Fig. 1 E ).…”

“…The nicotinamide amide nitrogen and oxygen atoms make hydrogen bonds to the Gly96 carbonyl oxygen and amide nitrogen atoms, respectively. The complementary hydrogen bonding of the nicotinamide amide to a conserved glycine in the HGT motif likely accounts for the inability of NAAD (nicotinic acid adenine dinucleotide) to serve as a substrate for Tpt1 in lieu of NAD + ( 11 ). In addition, the nicotinamide amide nitrogen makes an intramolecular hydrogen bond to a nonbridging NMN phosphate oxygen that aids in orienting the nicotinamide riboside ( Fig.…”

“…Fungal Tpt1 is viewed as a potential drug target because Tpt1 is essential for fungal growth ( 6 , 7 ) and the chemical mechanism of human tRNA splicing by GTP-dependent RtcB-type RNA ligase differs from that of fungi and does not result in a junction 2′-PO 4 ( 8 , 9 ). To provide tools for inhibitor discovery, Tpt1 orthologs have been characterized from four of the top-priority human fungal pathogens ( 10 ): Candida auris , Candida albicans , Aspergillus fumigatus , and Coccidioides immitis ( 11 ). Each of these pathogen Tpt1 enzymes can replace the essential Saccharomyces cerevisiae Tpt1 in vivo ( 11 ).…”

“…To provide tools for inhibitor discovery, Tpt1 orthologs have been characterized from four of the top-priority human fungal pathogens ( 10 ): Candida auris , Candida albicans , Aspergillus fumigatus , and Coccidioides immitis ( 11 ). Each of these pathogen Tpt1 enzymes can replace the essential Saccharomyces cerevisiae Tpt1 in vivo ( 11 ).…”