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, 39 (7), 2834-44

Evolution of Eukaryal tRNA-guanine Transglycosylase: Insight Gained From the Heterocyclic Substrate Recognition by the Wild-Type and Mutant Human and Escherichia Coli tRNA-guanine Transglycosylases

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Evolution of Eukaryal tRNA-guanine Transglycosylase: Insight Gained From the Heterocyclic Substrate Recognition by the Wild-Type and Mutant Human and Escherichia Coli tRNA-guanine Transglycosylases

Yi-Chen Chen et al. Nucleic Acids Res.

Abstract

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ(1) but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ(1). Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.

Figures

Scheme 1.
Scheme 1.
Synthesis of 1H- and 3H-labeled PreQ1 and Queuine: (a) trityl amine, Na2SO4, anhydrous THF, reflux, 6 h; (b) 2eq. NaBH4 or NaB3H4, THF, 0–25°C, 3 h, 79% two steps; (c) 1.25 M methanolic HCl, reflux, 2 h, 89%; (d) (3S,4R,5S)-3-amino-4,5-isopropylidenedioxy-cyclopentene, Na2SO4, MeOH, 25°C, 6 h (32); (e) 2eq. NaBH4 or NaB3H4, MeOH, 0–25°C, 4 h, 99% two steps (32); (f) 1.25 M methanolic HCl, 25°C, 2.5 h, 87% (32).
Figure 1.
Figure 1.
Selected regions of a sequence homology analysis of TGTs across the three kingdoms of life. A sequence alignment of representative TGTs from eubacteria, archaea and eukarya highlighting the conservation of aspartates 89, 143 and 264; cysteines 145, 302, 304 and 307; and histidine 333 (E. coli numbering). Dots indicate regions intentionally deleted for this figure. Dashes indicate gaps in the sequence alignment. The full alignment (see Supplementary Figure S17) was generated using PileUP in the SeqWeb software package (Accelerys). The conserved amino acids are colored-coded red.
Figure 2.
Figure 2.
Phylogenetic tree for TGTs across the three Kingdoms. The evolutionary history of the TGT enzymes was inferred via ‘maximum likelihood’ analysis with the MEGA4 software package. Representative TGT sequences spanning the three domains of life (13 eukaryal, 59 eubacterial and 72 archaeal) were globally aligned using Clustal W. The alignment was subjected to 500 bootstrap replicates resulting in the final consensus phylogenetic tree. This is an unrooted tree and the branch lengths are proportional to the evolutionary distances between nodes. Due to the number of sequences, a condensed version of the tree is shown. The full tree can be seen in Supplementary Figure S13.
Figure 3.
Figure 3.
Michaelis–Menten fits of (A) Queuine and (B) PreQ1 with wild-type human TGT.
Figure 4.
Figure 4.
Michaelis–Menten fit of PreQ1 with wild-type E. coli TGT.
Figure 5.
Figure 5.
Michaelis–Menten fit of PreQ1 with human TGT Val161Cys.
Figure 6.
Figure 6.
Active site of the PreQ1-bound Z. mobilis TGT (PDB accession code 1P0E). The (A) and (B) (view from two different angles) were generated by PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC). To be consistent with the text, the amino acid residues were labeled based on the E. coli numbering. Atoms oxygen, nitrogen and sulfur are highlighted in red, blue and yellow, respectively. Note: Tyr 93 in the Z. mobilis TGT corresponds to Phe 93 in the E. coli enzyme.

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