Figure 1.
Substrate bases of the Tgt enzymes from the three domains of life.
preQ0, 7-cyano-7-deazaguanine; preQ1, 7-aminomethyl-7-deazaguanine; queuine = 7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl) amino) methyl)-7-deazaguanine.
Figure 2.
Biosynthesis of the modified tRNA base queuine.
Substrates and cosubstrates: GTP, guanosine 5′-triphosphate; AdoMet, S-adenosylmethionine; B12, coenzyme B12; preQ0, 7-cyano-7-deazaguanine; preQ1, 7-aminomethyl-7-deazaguanine; enzymes: FolE, GTP cyclohydrolase I; QueD, 6-carboxy-5,6,7,8-tetrahydropterin synthase; QueE, 7-carboxy-7-deazaguanine synthase; QueC, preQ0 synthetase; QueF, preQ0 reductase; Tgt, tRNA-guanine transglycosylase; QueA, S-adenosylmethionine:tRNA ribosyltransferase-isomerase; QueG, epoxyqueuosine reductase.
Figure 3.
Assumed catalytic mechanism of bacterial Tgt.
(A and B) The glycosidic bond of guanosine 34 is cleaved via nucleophilic attack by the Asp280 carboxylate resulting in the formation of a covalent Tgt⋅tRNA intermediate. (C and D) Guanine is replaced by preQ1 which is incorporated into the tRNA via nucleophilic attack of the ribose 34 anomeric carbon by N9 of preQ1. Notably the replacement of guanine by preQ1 in the binding pocket of Tgt induces a flip of the Leu231/Ala232 peptide bond. The formation of a hydroxide and an oxonium ion as byproducts of the reaction is assumed to be responsible for its irreversibility as mutual neutralisation will efficiently detract these ions from equilibrium. H-bonds are indicated by dashed lines.
Figure 4.
Substrate base binding pocket of Z. mobilis Tgt and modelled human Tgt.
A) Detail of Z. mobilis Tgt·preQ1 complex crystal structure (PDB-code: 1p0e) showing the active site with the bound substrate in stick representation. Carbon atoms of protein residues are coloured in green, those of preQ1 in orange. B) Homology model of human Tgt created with the Z. mobilis Tgt crystal structure as a template. The close up shows active site residues (carbon atoms in grey) superimposed with preQ1 (carbon atoms in orange) as present in 1p0e. The coordinates of the homology model are provided within the Supporting Information (Coordinates S1).
Table 1.
Kinetic parameters for “wild type” Tgt and mutated Tgt variants.*
Figure 5.
Binding of tRNATyr to Tgt measured by Microscale Thermophoresis.
Alexa Fluor®647 labelled Tgt at a concentration of 50 nmol·L−1 was incubated with varying amounts of tRNATyr. The difference in normalised fluorescence as a measure of change in thermophoresis is plotted against the concentration of tRNATyr.
Figure 6.
Trapping experiments performed with Tgt/tRNA mixtures in presence of queuine or 2,6-diamino-3H-quinazolin-4-one.
A) Chemical structure of 2,6-diamino-3H-quinazolin-4-one (DAQ), an uncompetitive inhibitor of Tgt. B) SDS-PAGE analysis of reaction mixtures of Tgt or mutated variants thereof and tRNATyr under conditions indicated. SM, size marker; q, queuine. While DAQ causes retarded Tgt bands by stabilising the covalent Tgt·tRNA intermediate, queuine lacks this ability for most of the investigated Tgt variants. Solely in case of Tgt(Cys159Val/Val233Gly) a faint retarded band is visible indicating that queuine may to some extent be able to bind to the guanine 34/preQ1 subpocket of the covalent enzyme·tRNA complex.
Table 2.
Crystallographic data collection and refinement statistics - Tgt(Cys158Val).
Table 3.
Crystallographic data collection and refinement statistics – Tgt(Val233Gly).
Table 4.
Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.
Figure 7.
Crystal structures of Tgt variants in complex with preQ1 or queuine: surface representation of substrate pockets.
The solvent accessible surfaces of active sites from Z. mobilis Tgt variants are shown in bright yellow. The respective Tgt variant plus the bound ligand (shown in stick representation) are indicated in each sub-figure. Also amino acid residues at positions 158 and 233 are shown in stick representation. Carbon atoms of original amino acids and of the bound ligand are coloured green, those of mutated amino acids magenta. As the electron density assignable to the dihydroxy-cyclopentenyl moiety of queuine is poorly defined in all structures containing this ligand the coordinates of this moiety are not present in the respective structures deposited with the Protein Data Base. Accordingly, the conformations of the dihydroxy-cyclopentenyl shown in (D), (F) and (H) are tentative. To indicate this fact, the carbon atoms of this moiety are shown in grey. Selected water molecules are shown as red spheres. 2|Fo|-|Fc| (at σ 1.0) electron density is shown for the bound ligand and water molecules. 2|Fo|-|Fc| electron density contoured at a σ level of 1.0 is coloured blue, |Fo|-|Fc| electron density contoured at a σ level of 2.5 is coloured magenta. An overview of the crystal structures analysed in this study including resolutions and PDB codes is given in Table S1.
Figure 8.
Crystal structures of Tgt variants in complex with preQ1 or queuine: stick representation of substrate pockets.
The respective Tgt variant plus the bound ligand are indicated in each sub-figure. Carbon atoms of original amino acids are coloured green, those of mutated amino acids as well as of the bound ligand orange. As the electron density assignable to the dihydroxy-cyclopentenyl moiety of queuine is poorly defined in all structures containing this ligand the coordinates of this moiety are not present in the respective structures deposited with the Protein Data Base. Accordingly, the conformations of the dihydroxy-cyclopentenyl shown in (D), (F) and (H) are tentative. To indicate this fact, the carbon atoms of this moiety are shown in grey. Selected water molecules are shown as red spheres. Electron density is shown for amino acid residues at positions 158 and 233 as well as for the ligand and for water molecules. The 2|Fo|-|Fc| electron density map contoured at a σ level of 1.0 is coloured blue. The green density represents an |Fo|-|Fc| omit map contoured at 2.5 σ. An overview of the crystal structures analysed in this study including nominal resolutions and PDB codes is given in Table S1.
Figure 9.
Superimposition of preQ1-bound Tgt(Cys158Val) with its apo-, its guanine- and its queuine-bound form.
Carbon atoms of the Tgt(Cys158Val)·preQ1 complex are coloured pink in all sub-figures. (A) Superimposition (based on cα) of preQ1-bound Tgt(Cys158Val) and apo-Tgt(Cys158Val). Carbon atoms of apo-Tgt(Cys158Val) are coloured blue. Binding of preQ1 to Tgt(Cys158Val) provokes a shift of Val158 towards the ligand leading to the displacement of two water molecules (W1 and W2; shown as red spheres) which are present within this region in apo-Tgt(Cys158Val) and absent in the complex structure. In addition, the side chain of the proximate Thr159 rotates by about 90°. Exactly the same structural changes upon binding of preQ1 are observed for Tgt(Cys158Val/Val233Gly). (B) Superimposition (based on cα) of preQ1-bound Tgt(Cys158Val) and guanine-bound Tgt(Cys158Val). Carbon atoms of the Tgt(Cys158Val)·guanine complex are coloured yellow. In the Tgt(Cys158Val)·guanine complex, the side chain of Val158 becomes largely disordered. The Thr159 side chain adopts a similar conformation as observed in the apo-structure. (C) Superimposition (based on cα) of preQ1-bound Tgt(Cys158Val) and queuine-bound Tgt(Cys158Val). Carbon atoms of the Tgt(Cys158Val)·queuine complex are shown in grey. Binding of queuine obviously leads to disordering of the Val158 side chain as no electron density attributable to this isopropyl moiety is present in the electron density map of the refined Tgt(Cys158Val)·queuine complex structure. Also upon binding of queuine no conformational change of the Thr159 side chain is observed. It adopts a similar conformation as in the apo- and guanine-bound structures.
Table 5.
Oligonucleotides used in mutagenesis.