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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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Comparison of the surface models of YbeY and YfcM. (A) Ribbon and surface models of YbeY. The catalytic triad formed by three histidine residues (3-His motif) and Arg59 are depicted by ball-and-stick models. The putative catalytic cleft is depicted by an arrow. (B) Ribbon and surface models of YfcM.
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Figure 3: Comparison of the surface models of YbeY and YfcM. (A) Ribbon and surface models of YbeY. The catalytic triad formed by three histidine residues (3-His motif) and Arg59 are depicted by ball-and-stick models. The putative catalytic cleft is depicted by an arrow. (B) Ribbon and surface models of YfcM.

Mentions: A superimposition of the structures of YfcM and YbeY revealed that α2 and α3 of YfcM superimpose well on α5 and α6 of YbeY, respectively (Figure 2A, B and C). The root-mean-square (RMS) deviation between the residues from Tyr54 to Gly113, containing α2 and α3 of YfcM, and those from Leu104 to Leu144, containing α5 and α6 of YbeY, is 2.7 Å for all Cα atoms. The β1, β2 and β3 strands of YfcM correspond to the β2, β3 and β4 strands of YbeY, respectively, although the strand corresponding to β1 of YbeY is absent in YfcM (Figure 2A, B and C). The α-helix corresponding to α3 of YbeY does not exist in the structure of YfcM (Figure 2A, B and C). In YbeY, α3 forms the putative catalytic cleft, together with β3, α5 and the loop following α5 (41) (Figure 3A). The mutation of the highly conserved Arg59 on α3 generates severe defects in the ribonuclease activity of YbeY (41). The metal-binding three-histidine motif is also located on this cleft (Figure 3A). Due to the absence of the α-helix corresponding to α3 of YbeY, YfcM lacks this cleft (Figure 3B). The absence of the cleft is consistent with the fact that no ribonuclease activity has been reported for YfcM. On the other hand, YfcM harbors the C-terminal extension, consisting of α4, α5 and the following loop, which is absent in YbeY (Figure 2A, B and C). The C-terminal extension contacts α1, α2 and α3 and stabilizes the helices (Supplementary Figure S4A and B). Phe134 and Val138 on α4 hydrophobically interact with Val99 on α3, while Arg137 on α4 forms electrostatic contacts with Asp96 on α3 (Supplementary Figure S4A). Phe157 and Ala160 on α5 form a hydrophobic interaction network with Ile13, Phe14 and Phe18 on α1 and Ile61 and Trp64 on α2 (Supplementary Figure S4B). In addition, the side chain of Tyr165 on α5 forms a hydrogen bond with the side chain of Tyr54 on α2 (Supplementary Figure S4B). The stabilizing effect of the C-terminal extension is consistent with our observation that the deletion of the C-terminal extension of YfcM drastically reduces its solubility (data not shown). Overall, our structural data suggest that YfcM partially adopts the UPF0054 fold and, for the first time, provide evidence that the UPF0054 fold can act as hydroxylase.


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)

Comparison of the surface models of YbeY and YfcM. (A) Ribbon and surface models of YbeY. The catalytic triad formed by three histidine residues (3-His motif) and Arg59 are depicted by ball-and-stick models. The putative catalytic cleft is depicted by an arrow. (B) Ribbon and surface models of YfcM.
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Figure 3: Comparison of the surface models of YbeY and YfcM. (A) Ribbon and surface models of YbeY. The catalytic triad formed by three histidine residues (3-His motif) and Arg59 are depicted by ball-and-stick models. The putative catalytic cleft is depicted by an arrow. (B) Ribbon and surface models of YfcM.
Mentions: A superimposition of the structures of YfcM and YbeY revealed that α2 and α3 of YfcM superimpose well on α5 and α6 of YbeY, respectively (Figure 2A, B and C). The root-mean-square (RMS) deviation between the residues from Tyr54 to Gly113, containing α2 and α3 of YfcM, and those from Leu104 to Leu144, containing α5 and α6 of YbeY, is 2.7 Å for all Cα atoms. The β1, β2 and β3 strands of YfcM correspond to the β2, β3 and β4 strands of YbeY, respectively, although the strand corresponding to β1 of YbeY is absent in YfcM (Figure 2A, B and C). The α-helix corresponding to α3 of YbeY does not exist in the structure of YfcM (Figure 2A, B and C). In YbeY, α3 forms the putative catalytic cleft, together with β3, α5 and the loop following α5 (41) (Figure 3A). The mutation of the highly conserved Arg59 on α3 generates severe defects in the ribonuclease activity of YbeY (41). The metal-binding three-histidine motif is also located on this cleft (Figure 3A). Due to the absence of the α-helix corresponding to α3 of YbeY, YfcM lacks this cleft (Figure 3B). The absence of the cleft is consistent with the fact that no ribonuclease activity has been reported for YfcM. On the other hand, YfcM harbors the C-terminal extension, consisting of α4, α5 and the following loop, which is absent in YbeY (Figure 2A, B and C). The C-terminal extension contacts α1, α2 and α3 and stabilizes the helices (Supplementary Figure S4A and B). Phe134 and Val138 on α4 hydrophobically interact with Val99 on α3, while Arg137 on α4 forms electrostatic contacts with Asp96 on α3 (Supplementary Figure S4A). Phe157 and Ala160 on α5 form a hydrophobic interaction network with Ile13, Phe14 and Phe18 on α1 and Ile61 and Trp64 on α2 (Supplementary Figure S4B). In addition, the side chain of Tyr165 on α5 forms a hydrogen bond with the side chain of Tyr54 on α2 (Supplementary Figure S4B). The stabilizing effect of the C-terminal extension is consistent with our observation that the deletion of the C-terminal extension of YfcM drastically reduces its solubility (data not shown). Overall, our structural data suggest that YfcM partially adopts the UPF0054 fold and, for the first time, provide evidence that the UPF0054 fold can act as hydroxylase.

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.

Show MeSH
Related in: MedlinePlus