Seven-deazapurine modifications were thought to be highly specific of tRNAs, but have now been discovered in DNA of phages and of phylogenetically diverse bacteria, illustrating the plasticity of these modification pathways. The intermediate 7-cyano-7-deazaguanine (preQ₀) is a shared precursor in the pathways leading to the insetion of 7-deazapurine derivatives in both tRNA and DNA. It is also used as an intermediate in the synthesis of secondary metabolites such as toyocamacin. The presence of 7-deazapurine in DNA is proposed to be a protection mechanism against endonucleases. This makes preQ₀ a metabolite of underappreaciated but central importance.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.

The universal N(6)-threonylcarbamoyladenosine (t⁶A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t⁶A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t⁶A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t⁶A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB₂D₂), as well as 2:2:2 complex (TsaB₂D₂E₂), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB₂D₂ complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB₂D₂ alone, and a TsaB₂D₂E₁ complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t⁶A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t⁶A biosynthesis in bacteria.

Eukaryotic life benefits from—and ofttimes critically relies upon—the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5′-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.

The universally conserved N⁶-threonylcarbamoyladenosine (t⁶A) modification of tRNA is essential for translational fidelity. In bacteria, t⁶A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t⁶A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB₂D₂E₂) bound to Mg²⁺-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t⁶A cycle. Further, SAXS analysis of the TmTsaB₂D₂-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.