title: 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).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 co-expressed 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.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.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Chen
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Kelly
authors.forename: Stefanie V
authors.initials: SV
authors.lastname: Stachura
authors.forename: George A
authors.initials: GA
authors.lastname: Garcia
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits; D012365:RNA, Transfer, Tyr / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003432:Cross-Linking Reagents; D017931:DNA Primers; D021122:Protein Subunits; D012365:RNA, Transfer, Tyr; D011994:Recombinant Proteins; D006147:Guanine; C019549:queuine; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase