Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known. Here, we expand the knowledge of proteins isopeptide linked to SAMP1. LC-MS/MS analysis of -Gly-Gly signatures derived from SAMP1 S85R conjugates cleaved with trypsin was used to detect sites of sampylation (23 lysine residues) that mapped to 11 target proteins. Many of the identified target proteins were associated with sulfur metabolism and oxidative stress including MoaE, SAMP-activating E1 enzyme (UbaA), methionine sulfoxide reductase homologs (MsrA and MsrB), and the Fe-S assembly protein SufB. Several proteins were found to have multiple sites of samp1ylation, and the isopeptide linkage at SAMP3 lysines (K18, K55, and K62) revealed hetero-SAMP chain topologies. Follow-up affinity purification of selected protein targets (UbaA and MoaE) confirmed the LC-MS/MS results. 3D homology modeling suggested sampy1ylation is autoregulatory in inhibiting the activity of its protein partners (UbaA and MoaE), while occurring on the surface of some protein targets, such as SufB and MsrA/B. Overall, we provide evidence that SAMP1 is a ubiquitin-like protein modifier that is relatively specific in tagging its protein partners as well as proteins associated with oxidative stress response.

Pyrococcus horikoshii (Pho) RtcB exemplifies a family of binuclear transition metal- and GTP-dependent RNA ligases that join 3'-phosphate and 5'-OH ends via RtcB-(histidinyl-N)–GMP and RNA3'pp5'G intermediates. We find that guanylylation of PhoRtcB is optimal with manganese and less effective with cobalt and nickel. Zinc and copper are inactive and potently inhibit manganese-dependent guanylylation. We report crystal structures of PhoRtcB in complexes with GTP and permissive (Mn, Co, Ni) or inhibitory (Zn, Cu) metals. Zinc and copper occupy the M1 and M2 sites adjacent to the GTP phosphates, as do manganese, cobalt and nickel. The identity/positions of enzymic ligands for M1 (His234, His329, Cys98) and M2 (Cys98, Asp95, His203) are the same for permissive and inhibitory metals. The differences pertain to: (i) the coordination geometries and phosphate contacts of the metals; and (ii) the orientation of the His404 nucleophile with respect to the GTP α-phosphate and pyrophosphate leaving group. M2 metal coordination geometry correlates with metal cofactor activity, whereby inhibitory Zn2 and Cu2 assume a tetrahedral configuration and contact only the GTP γ-phosphate, whereas Mn2, Co2, and Ni2 coordination complexes are pentahedral and contact the β- and γ-phosphates. The His404-Nε–Pα–O(α-β) angle is closer to apical in Mn (179˚), Co (171˚), and Ni (169˚) structures than in Zn (160˚) and Cu (155˚) structures. The octahedral Mn1 geometry in our RtcB•GTP•Mn2+ structure, in which Mn1 contacts α-, β-, and γ-phosphates, transitions to a tetrahedral configuration after formation of RtcB•(His404)–GMP•Mn2+ and departure of pyrophosphate.

The enzyme Tpt1 removes an internal RNA 2 ′ ′ ′ ′ ′ -PO 4 via a two-step reaction in which: (i) the 2 ′ ′ ′ ′ ′ -PO 4 attacks NAD + to form an RNA-2 ′ ′ ′ ′ ′ -phospho-(ADP-ribose) intermediate and nicotinamide; 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 step 2 is much faster than step 1, the ADP-ribosylated RNA intermediate is virtually undetectable under normal circumstances. Here, by testing chemically modified nucleic acid substrates for activity with bacterial Tpt1 enzymes, we find that replacement of the ribose-2 ′ ′ ′ ′ ′ -PO 4 nucleotide with arabinose-2 ′ ′ ′ ′ ′ -PO 4 selectively slows step 2 of the reaction pathway and results in the transient accumulation of high levels of the reaction intermediate. We report that replacing the NMN ribose of NAD + with 2 ′ ′ ′ ′ ′ -fluoroarabinose (thereby eliminating the ribose O2 ′′ ′′ ′′ ′′ ′′ nucleophile) results in durable trapping of RNA-2 ′ ′ ′ ′ ′ -phospho-(ADP-fluoroarabinose) as a "dead-end" product of step 1. Tpt1 enzymes from diverse taxa differ in their capacity to use ara-2 ′′ ′′ ′′ ′′ ′′ F-NAD + as a substrate.

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.

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.