Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Abstract

The eukaryotic tRNA-guanine transglycosylase (TGT) has been reported to exist as a heterodimer, in contrast to the homodimeric eubacterial TGT. While ubiquitin-specific protease 14 (USP14) has been proposed to act as a regulatory subunit of the eukaryotic TGT, the mouse TGT has recently been shown to be a queuine tRNA-ribosyltransferase 1 (QTRT1, eubacterial TGT homolog) d queuine tRNA-ribosyltransferase domain-containing 1 (QTRTD1) heterodimer. We find that human QTRTD1 (hQTRTD1) co-purifies with polyhistidine-tagged human QTRT1 (ht-hQTRT1) via Ni 2+ affinity chromatography. Cross-linking experiments, mass spectrometry, and size exclusion chromatography results are consistent with the two proteins existing as a heterodimer. We have not been able to observe co-purification and/or association between hQTRT1 and USP14 when coexpressed in Escherichia coli. More importantly, under our experimental conditions, the transglycosylase activity of hQTRT1 is only observed when hQTRT1 and hQTRTD1 have been co-expressed and co-purified. Kinetic characterization of the human TGT (hQTRT1 d hQTRTD1) using human tRNA Tyr and guanine shows catalytic efficiency (k cat /K M ) similar to that of the E. coli TGT. Furthermore, site-directed mutagenesis confirms that the hQTRT1 subunit is responsible for the transglycosylase activity. Taken together, these results indicate that the human TGT is composed of a catalytic subunit, hQTRT1, and hQTRTD1, not USP14. hQTRTD1 has been implicated as the salvage enzyme that generates free queuine from QMP. Work is ongoing in our laboratory to confirm this activity.

Plant, Animal, and Fungal Micronutrient Queuosine Is Salvaged by Members of the DUF2419 Protein Family

“…The known and published sequences of the characterized bacterial TGT ( E. coli TGT, AAA24667 68 ), and human QTRT1, and QTRTD1 (IPI00215974.2 and IPI00783033.2, respectively 17 ) were used as entry points for all database queries. The BLAST tools 69 and resources at NCBI (National Center for Biotechnology Information, ) were routinely used.…”

Biosynthesis of pyrrolopyrimidines

“…QTRT1 associates with another protein, QTRTD1, which is a splice variant of QTRT1 and shares significant sequence identity with it; the two associate in vivo and appear to localize to the outer membrane of the mitochondria [84]. Complex formation, either by co-expression [84] or in vitro mixing [85] leads to active protein. The bacterial and mammalian proteins have been proposed to have evolved via divergent evolution, and there is evidence that small changes in active site architecture are responsible for their differential substrate recognition properties [86,87].…”

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification

“…Q modification is observed both on the cytosolic and the mitochondrial variant of these tRNAs [13,27]. The eTGTs consist of two subunits that both are evolutionarily related to bTGT: the catalytic subunit queuine-tRNA-ribosyltransferase (QTRT1) and the accessory subunit queuine-tRNA ribosyltransferase domain containing 1 (QTRTD1, re-annotated as QTRT2) [28]. It is interesting to note that eTGT acts as a heterodimer, whereas bTGT functions as a homodimer [29].…”