Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

Abstract

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an alpha-amino-alpha-carboxypropyl ("acp") group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the "acp" group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNA(Phe), and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this "acp" group has a significant role in preventing decoding frame shifts, the mechanism of the "acp" group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the "acp" group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the "acp" group, and not the methyl group, from AdoMet to the nucleobase.

Fig. 1.

Acp-group transfer catalyzed by PhTYW2. (A) Biosynthetic pathway of wybutosine. The chemical structures of the wyosine derivatives are shown. Modification enzymes, Trm5 and TYW1–4, are involved in each modification step. The chemical groups included in each reaction are shadowed in blue. The chemical structure of AdoMet is shown in the box. The methyl and acp groups are colored green and blue, respectively. (B) In vitro reconstitution of yW-86 synthesis using recombinant PhTYW2. RNaseT₁-digested yeast tRNA^Phe with the imG-14 intermediate (black), obtained from the ΔTYW2 strain, was reacted with PhTYW2 in the presence (left panel) or absence (right panel) of AdoMet and was analyzed by LC/MS. Mass chromatographs for anticodon-containing fragments including imG-14 (m/z 661, black), yW-86 (m/z 678, green), and yW-173 (m/z 664, red) are overlaid. (C) Kinetic analysis of the acp-group transfer by PhTYW2. The α-carboxyl-¹⁴C labeled AdoMet and the imG-14 containing tRNA^Phe were incubated at 50 °C with recombinant PhTYW2 (closed circles) and without PhTYW2 (open circles), and the incorporation of α-carboxyl-¹⁴C into the tRNA^Phe was quantified by scintillation counting. Data are represented as mean ± SE (n = 3).

Fig. 2.

Overall structure of archaeal TYW2. (A) Crystal structure of AdoMet-bound PhTYW2. (B) Crystal structure of AdoMet-bound MjTYW2. In (A and B), the N-terminal and C-terminal domains are colored green and blue, respectively. AdoMet is shown in a stick model. The disordered regions are depicted by dashed lines. 2F_o−F_c annealed omit maps around AdoMet contoured at 1.0 σ are shown. (C) Topology diagram of the PhTYW2 structure with the same color code as in (A). The PhTYW2-specific extension region is enclosed by a dashed circle, and the disordered region is indicated by dashed lines. The AdoMet-binding site is indicated with a circle.

Fig. 3.

Structural comparison of the cofactor-binding pockets in (A) PhTYW2 with AdoMet and (B) Trm5 with sinefungine (SFG) (PDB ID 2YX1). AdoMet and SFG are colored orange and light green, respectively. Residues involved in the cofactor recognition are shown by stick models (left panels). Interactions are indicated with black dashed lines. Motifs I, II, and III are colored green, yellow, and cyan, respectively. His-138 (A) and Pro-208 (B) are colored blue, and the hydrophobic residues that recognize the adenine moiety of AdoMet are colored gray. Schematic diagrams of the cofactor binding pocket are shown in the right panels. The M and A cavities are shown in green and magenta circles, respectively. In the schematic diagram of (B), AdoMet is shown instead of the inhibitor SFG.

Fig. 4.

Acp-group transfer activity of the wild-type and mutants of PhTYW2. The relative activities to the wild-type enzyme were evaluated from the initial velocities calculated from data in Fig. S5B.

Fig. 5.

Structural comparison of TYW2 and Trm5. (A) Comparison of the M cavities of PhTYW2 and MjTrm5. (B) Comparison of the A cavities of PhTYW2 and MjTrm5. PhTYW2-AdoMet and Trm5-SFG (PDB ID 2YX1) are colored gray and green, respectively. The M and A cavities are enclosed by dashed circles in panels (A and B), respectively.

Fig. 6.

tRNA^Phe and imG-14 recognition mechanism by PhTYW2. (A) Overview of the Trm5·tRNA^Cys complex structure. (B) Overview of the PhTYW2·tRNA docking model. The tRNA molecules are colored pink. The electrostatic potential distribution is presented on the contact surface of PhTYW2, with positively and negatively charged regions colored blue and red, respectively. The contact surface and the electrostatic potential were calculated by PyMOL (http://pymol.sourceforge.net/). (C and D) Active sites of the Trm5·tRNA^Cys complex and the PhTYW2 docking model. The G37 and imG-14 residues and AdoMet are shown by stick models. Hydrogen bonds are indicated by black dashed lines. The acceptor atoms of the chemical groups transferred from AdoMet are indicated by arrows.