The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the
unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its
ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we
confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a
conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian
tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts
RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an
antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions.
Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus,
we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein
PYROXD1 through an unexpected redox mechanism