Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase

Abstract

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis–Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

Translational response to mitochondrial stresses is orchestrated by tRNA modifications

“…LC-MS/MS and codon analysis data strongly pointed to tRNA queuosine (tRNA-Q) as a major regulator of the translational response to mitochondrial stress and dysfunction. tRNA-Q is synthesized by tRNA-guanine trans-glycosylase (TGT) complex, which is composed of 2 enzymes: QTRT1 and QTRT2(Behrens et al, 2018; Johannsson et al, 2018; Sebastiani et al, 2022; Sievers et al, 2021). The TGT complex utilizes Queuine to convert Guanosine into Queosine in GUN anticodon, which is essential for the expanding the codon recognition from NAC codons to NAY codons(Dixit et al, 2021; Huber et al, 2022; Sebastiani et al, 2022; Suzuki, 2021; Tuorto et al, 2018) [Figure 5a].…”

“…tRNA-Q is synthesized by tRNA-guanine trans-glycosylase (TGT) complex, which is composed of 2 enzymes: QTRT1 and QTRT2(Behrens et al, 2018; Johannsson et al, 2018; Sebastiani et al, 2022; Sievers et al, 2021). The TGT complex utilizes Queuine to convert Guanosine into Queosine in GUN anticodon, which is essential for the expanding the codon recognition from NAC codons to NAY codons(Dixit et al, 2021; Huber et al, 2022; Sebastiani et al, 2022; Suzuki, 2021; Tuorto et al, 2018) [Figure 5a]. However, QTRT1 and QTRT2 protein levels did not change after 8 hours of stress [Supplementary figure 12a], contrary to the strong upregulation of queuosine levels in tRNA.…”

Covalent Catalytic Strategies for Enzymes That Modify RNA Molecules on their Tripartite Building Blocks

“…The enzyme responsible for removing guanine and replacing it with the 7-deazaguanine queuosine is a tRNA glycosyltransferase (tRNA guanine transglycosylase). 42 As the enzyme name implies, this enzyme works on the glycosyl group. In the case of nucleoside monophosphate (NMP) building blocks in RNAs, the glycosyl group is the ribofuranose ring.…”

“…Eukaryotes swap in queuine directly into the tRNAs. The enzyme responsible for removing guanine and replacing it with the 7-deazaguanine queuosine is a tRNA glycosyltransferase (tRNA guanine transglycosylase) . As the enzyme name implies, this enzyme works on the glycosyl group.…”

Queuine Analogues Incorporating the 7‐Aminomethyl‐7‐deazaguanine Core: Structure–Activity Relationships in the Treatment of Experimental Autoimmune Encephalomyelitis

“…[9,10] This unique reaction is catalysed by the queuine tRNA ribosyltransferase (QTRT) enzyme -also frequently referred to as tRNA guanine transglycosylase (TGT, 5-a heterodimeric complex of the QTRT1 and QTRT2 proteins [11] ; both of which are required for biological activity). [12,13] While QTRT can also mediate a guanine-forguanine exchange reaction, the installation of queuine is essentially irreversible. [14] An analogous process in eubacteria equips the appropriate tRNAs 1 with a simplified queuine analogue (preQ 1 ) at position 34, with subsequent enzymatic elaboration to 4.…”