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The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation.

Kobayashi K, Katz A, Rajkovic A, Ishii R, Branson OE, Freitas MA, Ishitani R, Ibba M, Nureki O - Nucleic Acids Res. (2014)

Bottom Line: The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology.Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the β-lysylated lysine residue of EF-P.Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Global Research Cluster, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan.

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Co(II) ion coordination by YfcM. The 2Fo-Fc electron density map contoured at 1.0σ and the anomalous difference Fourier map contoured at 20σ are shown in blue and red, respectively. The bound Co(II) ion and water molecules are depicted by gray and red spheres, respectively. Residues involved in the coordination of the Co(II) ion are depicted by ball-and-stick models.
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Figure 5: Co(II) ion coordination by YfcM. The 2Fo-Fc electron density map contoured at 1.0σ and the anomalous difference Fourier map contoured at 20σ are shown in blue and red, respectively. The bound Co(II) ion and water molecules are depicted by gray and red spheres, respectively. Residues involved in the coordination of the Co(II) ion are depicted by ball-and-stick models.

Mentions: We tested whether an Fe ion binds to YfcM. The purified YfcM protein was mixed with FeSO4, and the free FeSO4 was removed by gel-filtration. The fraction containing YfcM was analyzed by atomic absorption spectrometry. The results showed that an Fe ion coeluted with YfcM from the gel-filtration column, and the molar ratio of YfcM and Fe was 1 : 0.37 (Fe-bound, Supplementary Table S3). Therefore, the Fe ion directly binds to YfcM. In contrast, an Fe ion was not observed in the purified YfcM in the absence of exogenous Fe ion (Apo, Supplementary Table S3). This may be because the Fe(II) ion bound to YfcM was oxidized to an Fe(III) ion during the purification process. The ionic radius change would destabilize the interaction between the Fe(III) ion and YfcM, leading to the dissociation of the Fe(III) ion from YfcM. The metal ion observed in the structure of YfcM could be another metal ion, such as Mg(II), which may have bound during the purification process. In order to confirm that an Fe(II) ion is coordinated by the putative 2-His-1-carboxylate motif of YfcM, we prepared crystals of Co(II)-bound YfcM and calculated the anomalous difference Fourier map, using the data set collected at the peak wavelength of the Co K-shell absorption edge (1.6049 Å). The Co(II) ion has an ionic radius similar to that of an Fe(II) ion (43) and can adopt the geometry of an octahedral complex together with six ligands, similar to an Fe(II) ion (43). Therefore, a Co(II) ion often binds to the 2-His-1-carboxylate motif in a manner that mimics an Fe(II) ion. In fact, it was reported that the substitution of the Fe(II) ion for the Co(II) ion in the 2-His-1-carboxylate motif of homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has almost no effect on the arrangement of metal ion-coordinating residues and the overall structure of HPCD (44). Furthermore, a Co(II) ion occupies an Fe(II) ion-coordinating site more stably than an Fe(II) ion, because an Fe(II) ion is easily air-oxidized to an Fe(III) ion, thus changing its ionic radius. The resultant electron density map clearly exhibited a strong Co(II) peak at the metal-coordinating site, indicating that a Co(II) ion can be coordinated by His59, His63 and Glu98 (Figure 5). In this structure, six atoms participate in the Co(II) ion coordination (nitrogen atoms (NEs) of His59 and His63, two oxygen atoms (OEs) of Glu98 and two oxygen atoms of two water molecules; distances from the Co(II) ion are summarized in Supplementary Table S5), which may mimic the Fe(II) ion coordination (Figure 5). This observation further indicated that His59, His63 and Glu98 can coordinate an Fe(II) ion. Therefore, our data suggested that His59, His63 and Glu98 of YfcM form the putative 2-His-1-carboxylate motif for the coordination of an Fe(II) ion, as in the active sites of non-heme iron enzymes.


The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation.

Kobayashi K, Katz A, Rajkovic A, Ishii R, Branson OE, Freitas MA, Ishitani R, Ibba M, Nureki O - Nucleic Acids Res. (2014)

Co(II) ion coordination by YfcM. The 2Fo-Fc electron density map contoured at 1.0σ and the anomalous difference Fourier map contoured at 20σ are shown in blue and red, respectively. The bound Co(II) ion and water molecules are depicted by gray and red spheres, respectively. Residues involved in the coordination of the Co(II) ion are depicted by ball-and-stick models.
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Figure 5: Co(II) ion coordination by YfcM. The 2Fo-Fc electron density map contoured at 1.0σ and the anomalous difference Fourier map contoured at 20σ are shown in blue and red, respectively. The bound Co(II) ion and water molecules are depicted by gray and red spheres, respectively. Residues involved in the coordination of the Co(II) ion are depicted by ball-and-stick models.
Mentions: We tested whether an Fe ion binds to YfcM. The purified YfcM protein was mixed with FeSO4, and the free FeSO4 was removed by gel-filtration. The fraction containing YfcM was analyzed by atomic absorption spectrometry. The results showed that an Fe ion coeluted with YfcM from the gel-filtration column, and the molar ratio of YfcM and Fe was 1 : 0.37 (Fe-bound, Supplementary Table S3). Therefore, the Fe ion directly binds to YfcM. In contrast, an Fe ion was not observed in the purified YfcM in the absence of exogenous Fe ion (Apo, Supplementary Table S3). This may be because the Fe(II) ion bound to YfcM was oxidized to an Fe(III) ion during the purification process. The ionic radius change would destabilize the interaction between the Fe(III) ion and YfcM, leading to the dissociation of the Fe(III) ion from YfcM. The metal ion observed in the structure of YfcM could be another metal ion, such as Mg(II), which may have bound during the purification process. In order to confirm that an Fe(II) ion is coordinated by the putative 2-His-1-carboxylate motif of YfcM, we prepared crystals of Co(II)-bound YfcM and calculated the anomalous difference Fourier map, using the data set collected at the peak wavelength of the Co K-shell absorption edge (1.6049 Å). The Co(II) ion has an ionic radius similar to that of an Fe(II) ion (43) and can adopt the geometry of an octahedral complex together with six ligands, similar to an Fe(II) ion (43). Therefore, a Co(II) ion often binds to the 2-His-1-carboxylate motif in a manner that mimics an Fe(II) ion. In fact, it was reported that the substitution of the Fe(II) ion for the Co(II) ion in the 2-His-1-carboxylate motif of homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has almost no effect on the arrangement of metal ion-coordinating residues and the overall structure of HPCD (44). Furthermore, a Co(II) ion occupies an Fe(II) ion-coordinating site more stably than an Fe(II) ion, because an Fe(II) ion is easily air-oxidized to an Fe(III) ion, thus changing its ionic radius. The resultant electron density map clearly exhibited a strong Co(II) peak at the metal-coordinating site, indicating that a Co(II) ion can be coordinated by His59, His63 and Glu98 (Figure 5). In this structure, six atoms participate in the Co(II) ion coordination (nitrogen atoms (NEs) of His59 and His63, two oxygen atoms (OEs) of Glu98 and two oxygen atoms of two water molecules; distances from the Co(II) ion are summarized in Supplementary Table S5), which may mimic the Fe(II) ion coordination (Figure 5). This observation further indicated that His59, His63 and Glu98 can coordinate an Fe(II) ion. Therefore, our data suggested that His59, His63 and Glu98 of YfcM form the putative 2-His-1-carboxylate motif for the coordination of an Fe(II) ion, as in the active sites of non-heme iron enzymes.

Bottom Line: The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology.Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the β-lysylated lysine residue of EF-P.Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Global Research Cluster, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan.

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Related in: MedlinePlus