Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit

Deshpande, Kathryn L.; Katze, Jon R.

doi:10.1016/s0378-1119(01)00368-7

Cited by 12 publications

(13 citation statements)

References 17 publications

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“…However, no crystal structure of either of these enzymes is available yet, and the only structural information concerning the active site of a eucaryotic Tgt originates from a homology model of the Caenorhabditis elegans catalytic subunit [35]. As the complete sequence of the human catalytic subunit has meanwhile become available [28], [36] we created a model of this orthologue using the crystal structure of Z. mobilis Tgt as a template (for experimental details see the “Materials and Methods” section). The model indicates that most residues establishing the active centre and substrate binding pocket in the bacterial and human enzyme are identical.…”

Section: Resultsmentioning

confidence: 99%

Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

et al. 2013

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Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAsHis,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering) by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

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Section: Resultsmentioning

confidence: 99%

Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

et al. 2013

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“…Initial searches identified a mouse TGT (NM021888), transcribed from the queuine tRNA ribosyltransferase (QTRT1) gene on chromosome 9, which is equivalent to the previously identified human TGT catalytic subunit (21). Further searches, using both the E. coli and mouse TGT proteins as bait, revealed two additional related mouse members of the family (NM029128 and BC017628), and a third was subsequently identified in male mouse kidney (FM985972) by RT-PCR (see "Experimental Procedures").…”

Section: Eukaryotes Contain Two Genes Whose Productsmentioning

confidence: 99%

“…Indeed, it has been stated that the cDNA for the human TGT catalytic subunit is toxic to E. coli (21), and it has been shown that expression of E. coli TGT in a naturally Q-deficient E. coli stain, B105, results in loss of viability (30).…”

Section: Interaction Of Tgt and Qv1 Is Required For Trna Guaninementioning

confidence: 99%

Queuosine Formation in Eukaryotic tRNA Occurs via a Mitochondria-localized Heteromeric Transglycosylase

Boland

Hayes

Santa-María

et al. 2009

Journal of Biological Chemistry

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tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA Asp , tRNA Asn , tRNA His , and tRNA Tyr ; an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle. Queuosine (Q 3 ; (7-{[(4,5-cis-dihydroxy-2-cyclo-penten-1-yl)-amino]methyl}-7-deazaguanosine) is a modified 7-deazaguanosine molecule found at the wobble position of transfer RNA that contains a GUN anticodon sequence: tRNA Tyr , tRNA Asn , tRNA His , and tRNA Asp (1). The Q-modification is widely distributed in nature in the tRNA of eubacteria, plants, and animals; a notable exception being yeast and plant leaf cells (2, 3). Interestingly, Q-modification has also been detected in aspartyl tRNA from mitochondria of rat (4) and opossum (5). In most eukaryotes, the Q molecule can be further modified by the addition of a mannosyl group to Q-tRNA Asp and a galactosyl group to Q-tRNA Tyr (1). Eubacteria are unique in their ability to synthesize Q. As part of this biosynthetic process, the eubacterial tRNA guanine transglycosylase (TGT) enzyme inserts the Q precursor molecule, 7-aminomethyl-7-deazaguanine (preQ 1 ) into tRNA, which is then converted to Q by two further enzymatic steps at the tRNA level (6). Eukaryotes by contrast salvage queuosine from food and enteric bacteria either as the free base (referred to as queuine) or as queuosine 5Ј-phosphate subsequent to normal tRNA turnover (7). A Q-related molecule, archaeosine, is found at position 15 of the D loop of most archaeal tRNA, where it functions to stabilize the tRNA structure (8). The enzyme involved in archaeosine biosynthesis is structurally and mechanistically related to the eubacterial TGT but with adaptations necessitated by the differences imposed by its unique substrate and tRNA specificity (9, 10).The crystal structure of the Zymononas mobilis (Z. mobilis) TGT has been determined and revealed the enzyme to be an irregular (␤/␣) 8 TIM barrel with a C-terminal zinc-binding subdomain (11). Insight into the residues involved in catalysis came from mutational and kinetic analysis of the recombinant Escherichia coli enzyme...

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“…The queuine tRNA-ribosyltransferase 1 (qtrt1) gene was identified as the catalytic subunit due to a high degree of protein sequence homology (z40%) to the eubacterial TGT. The 60-kDa subunit has no TGT-like catalytic activity and has been annotated as a member of the ubiquitin-specific protease family (USP14) (Deshpande et al 1996;Deshpande and Katze 2001). Morris et al (1995) reported that the 60-kDa subunit of rat liver TGT (presumably USP14) is a protein kinase C (PKC) substrate and proposed that unphosphorylated TGT exists in a low-activity, dimeric state.…”

Section: Introductionmentioning

confidence: 99%

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

Chen

Kelly

Stachura

et al. 2010

RNA

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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.

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Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit

Cited by 12 publications

References 17 publications

Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

Queuosine Formation in Eukaryotic tRNA Occurs via a Mitochondria-localized Heteromeric Transglycosylase

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

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