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.