Evolution of Eukaryal tRNA-Guanine Transglycosylase: Insight Gained from the Characterization of the Human and Escherichia coli tRNA-Guanine Transglycosylases.

Human TRNA-guanine Transglycosylase;Queuosine Modification;Heterodimeric Subunit Structure;Divergent Evolution of TGT;Characterization of the Eukaryal TGT;Biological Chemistry;Pharmacy and Pharmacology;Health Sciences;Medicinal Chemistry

Chen, Yi-ChenWoodard, Ronald W. ;

University of Michigan

关键词: Human TRNA-guanine Transglycosylase;    Queuosine Modification;    Heterodimeric Subunit Structure;    Divergent Evolution of TGT;    Characterization of the Eukaryal TGT;    Biological Chemistry;    Pharmacy and Pharmacology;    Health Sciences;    Medicinal Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/84565/yichenc_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Of the approximately 100 tRNA modifications that have been identified thus far, queuine (Q, 7-((4, 5-cis-dihydroxy-2-cyclopenten-1-yl) amino) methyl-7-deazaguanine) is one of the most complicated.Although its physiological role is largely obscure, the Q modification of tRNA in eukarya has been suggested to be involved in cell differentiation, proliferation and response to oxidative stress.Additionally, the malfunction of Q modification has been correlated with several biological disorders.These observations have elicited an interest in acquiring a greater understanding of the physiological role(s) of the queuosine modification.To explore the biological significance of queuosine modification in eukarya, this dissertation work studied the key human enzyme, tRNA-guanine transglycosylase (TGT), which catalyzes the exchange of queuine for guanine inits substrate tRNAs.The eukaryal TGT was previously proposed to be a heterodimeric protein composed of queuine tRNA-ribosyltransferase 1 (QTRT1) and ubiquitin-specific protease 14 (USP14).However, physical and kinetic evidence that we have obtained make it clear that the human TGT consists of QTRT1 and its homologue, queuine tRNA-ribosyltransferase domain-containing 1 (QTRTD1).Additional mutagenesis studies reveal that QTRT1 is responsible for the transglycosylase activity.Our results along with previous observations by others also led us to propose that QTRTD1 not only assists in recognizing substrate tRNAs, but has also evolved into a queuine salvage enzyme that generates the heterocyclic substrate (queuine) for the transglycosylation.Future work is pending to validate the latter part of this hypothesis.To the best of our knowledge, we have also provided the first report of the kinetics of the eukaryal TGT via direct incorporation assays.In vitro kinetic studies reported in this dissertation demonstrate preferential heterocyclic substrate recognition for the human TGT.Follow-up work of wild-type and mutant, human and Escherichia coli TGTs confirmed that active site residues of both enzymes evolved to selectively recognize their cognate heterocyclic substrates.This work has provided a greater understanding of the eukaryal TGT via rigorous enzymatic characterization of the recombinant human and E. coli TGTs.Our results are consistent with the concept that the present forms of TGT have arisen via divergent evolution.