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Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification.

Kato M, Araiso Y, Noma A, Nagao A, Suzuki T, Ishitani R, Nureki O - Nucleic Acids Res. (2010)

Bottom Line: Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations.Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown.These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.

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The active site and catalytic mechanism of TYW5. (A) An unbiased Fo – Fc omit map around 2-OG (contoured at 3.0σ) and an anomalous Fourier map around the nickel ion (contoured at 20.0σ) are shown in blue and red, respectively. The omit map was calculated after removing the 2-OG cofactor from the model. (B) The active site of FIH complexed with the substrate peptide. The same color code as in Figure 3B is used. The substrate peptide of FIH is colored red. FIH catalyzes the hydroxylation of the β-carbon of Asn803, which is encircled with the β carbon indicated by an arrow. (C) Proposed catalytic mechanism by hTYW5.
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Figure 4: The active site and catalytic mechanism of TYW5. (A) An unbiased Fo – Fc omit map around 2-OG (contoured at 3.0σ) and an anomalous Fourier map around the nickel ion (contoured at 20.0σ) are shown in blue and red, respectively. The omit map was calculated after removing the 2-OG cofactor from the model. (B) The active site of FIH complexed with the substrate peptide. The same color code as in Figure 3B is used. The substrate peptide of FIH is colored red. FIH catalyzes the hydroxylation of the β-carbon of Asn803, which is encircled with the β carbon indicated by an arrow. (C) Proposed catalytic mechanism by hTYW5.

Mentions: In the crystal structure of the cofactor-bound TYW5, we observed a strong peak in the anomalous difference Fourier map, calculated from the dataset collected at the peak wavelength of the Ni K-shell absorption edge. Therefore, we assigned this peak to the Ni(II) ion bound to the catalytic site (Figure 4A). As in the typical Fe(II)/2-OG oxygenases, the Ni(II) ion is coordinated by the signature motif of His160, Asp162 (on the loop connecting strands β7 and β9) and His235 (on β13), and mimics the physiological cofactor, Fe(II) ion (Figure 4A). Furthermore, in this cofactor-bound form, we clearly observed an electron density peak corresponding to the 2-OG cofactor (Figure 4A). The bound 2-OG cofactor is recognized by the conserved residues among TYW5 and FIH in the catalytic pocket, where the ε-amino group of Lys175 and the hydroxyl group of Tyr106 hydrogen bond to the C-5 carboxyl moiety of 2-OG, and the side chain amino group of Asn166 hydrogen bonds to the C-1 carboxyl moiety of 2-OG (Figure 4A). The side-chain conformations of these residues in hTYW5, as well as the 2-OG recognition manner, are also similar to those in FIH.Figure 4.


Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification.

Kato M, Araiso Y, Noma A, Nagao A, Suzuki T, Ishitani R, Nureki O - Nucleic Acids Res. (2010)

The active site and catalytic mechanism of TYW5. (A) An unbiased Fo – Fc omit map around 2-OG (contoured at 3.0σ) and an anomalous Fourier map around the nickel ion (contoured at 20.0σ) are shown in blue and red, respectively. The omit map was calculated after removing the 2-OG cofactor from the model. (B) The active site of FIH complexed with the substrate peptide. The same color code as in Figure 3B is used. The substrate peptide of FIH is colored red. FIH catalyzes the hydroxylation of the β-carbon of Asn803, which is encircled with the β carbon indicated by an arrow. (C) Proposed catalytic mechanism by hTYW5.
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Figure 4: The active site and catalytic mechanism of TYW5. (A) An unbiased Fo – Fc omit map around 2-OG (contoured at 3.0σ) and an anomalous Fourier map around the nickel ion (contoured at 20.0σ) are shown in blue and red, respectively. The omit map was calculated after removing the 2-OG cofactor from the model. (B) The active site of FIH complexed with the substrate peptide. The same color code as in Figure 3B is used. The substrate peptide of FIH is colored red. FIH catalyzes the hydroxylation of the β-carbon of Asn803, which is encircled with the β carbon indicated by an arrow. (C) Proposed catalytic mechanism by hTYW5.
Mentions: In the crystal structure of the cofactor-bound TYW5, we observed a strong peak in the anomalous difference Fourier map, calculated from the dataset collected at the peak wavelength of the Ni K-shell absorption edge. Therefore, we assigned this peak to the Ni(II) ion bound to the catalytic site (Figure 4A). As in the typical Fe(II)/2-OG oxygenases, the Ni(II) ion is coordinated by the signature motif of His160, Asp162 (on the loop connecting strands β7 and β9) and His235 (on β13), and mimics the physiological cofactor, Fe(II) ion (Figure 4A). Furthermore, in this cofactor-bound form, we clearly observed an electron density peak corresponding to the 2-OG cofactor (Figure 4A). The bound 2-OG cofactor is recognized by the conserved residues among TYW5 and FIH in the catalytic pocket, where the ε-amino group of Lys175 and the hydroxyl group of Tyr106 hydrogen bond to the C-5 carboxyl moiety of 2-OG, and the side chain amino group of Asn166 hydrogen bonds to the C-1 carboxyl moiety of 2-OG (Figure 4A). The side-chain conformations of these residues in hTYW5, as well as the 2-OG recognition manner, are also similar to those in FIH.Figure 4.

Bottom Line: Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations.Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown.These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.

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