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.