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Predicting the pathway involved in post-translational modification of elongation factor P in a subset of bacterial species.

Bailly M, de Crécy-Lagard V - Biol. Direct (2010)

Bottom Line: Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway.Zhulin and Mikhail Gelfand.For the full reviews, please go to the Reviewers' reports section.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA.

ABSTRACT

Background: The bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for protein synthesis. It is homologous to the eukaryotic translation initiation factor 5A (eIF5A). A highly conserved eIF5A lysine is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for eIF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a spermidine derivative in Escherichia coli. However, the biosynthesis pathway of this modification in the bacterial EF-P is yet to be elucidated.

Presentation of the hypothesis: Here we propose a potential mechanism for the post-translational modification of EF-P. By using comparative genomic methods based on physical clustering and phylogenetic pattern analysis, we identified two protein families of unknown function, encoded by yjeA and yjeK genes in E. coli, as candidates for this missing pathway. Based on the analysis of the structural and biochemical properties of both protein families, we propose two potential mechanisms for the modification of EF-P.

Testing the hypothesis: This hypothesis could be tested genetically by constructing a bacterial strain with a tagged efp gene. The tag would allow the purification of EF-P by affinity chromatography and the analysis of the purified protein by mass spectrometry. yjeA or yjeK could then be deleted in the efp tagged strain and the EF-P protein purified from each mutant analyzed by mass spectrometry for the presence or the absence of the modification. This hypothesis can also be tested by purifying the different components (YjeK, YjeA and EF-P) and reconstituting the pathway in vitro.

Implication of the hypothesis: The requirement for a fully modified EF-P for protein synthesis in certain bacteria implies the presence of specific post-translational modification mechanism in these organisms. All of the 725 bacterial genomes analyzed, possess an efp gene but only 200 (28%) possess both yjeA and yjeK genes. In the other organisms, EF-P may be modified by another pathway or the translation machinery must have adapted to the lack of EF-P modification. Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway.

Reviewers: This article was reviewed by Céline Brochier-Armanet, Igor B. Zhulin and Mikhail Gelfand. For the full reviews, please go to the Reviewers' reports section.

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Potential EF-P modification pathways. A- Mechanism in which YjeA acts first on free lysine (Lys) and attaches it to EF-P Lys34 which is then modified on EF-P into β-lysine by YjeK. B- Mechanism in which YjeK acts first to modify free lysine into β-lysine which is subsequently activated by YjeA and attached to EF-P lysine 34. EF-P N-terminal loop is indicated in yellow, Lys 34 is indicated in red, the modification appear on light brown and the AMP generated by YjeA during the activation of the Lys residue appear in blue. Potential and known substrates and cofactors of each enzyme are indicated.
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Figure 4: Potential EF-P modification pathways. A- Mechanism in which YjeA acts first on free lysine (Lys) and attaches it to EF-P Lys34 which is then modified on EF-P into β-lysine by YjeK. B- Mechanism in which YjeK acts first to modify free lysine into β-lysine which is subsequently activated by YjeA and attached to EF-P lysine 34. EF-P N-terminal loop is indicated in yellow, Lys 34 is indicated in red, the modification appear on light brown and the AMP generated by YjeA during the activation of the Lys residue appear in blue. Potential and known substrates and cofactors of each enzyme are indicated.

Mentions: In the first model (Fig. 4A), YjeA activates (S)-α-lysine into (S)-α-lysyl•AMP in presence of ATP and magnesium. This is coherent with the presence of AMP in the YjeA three dimensional structure (PDB: 3G1Z) and the conservation of the lysine binding residues in the YjeA active site (Fig. 3C). The activated (S)-α-lysine could then be transferred to the conserved Lys34 of EF-P to form an iso-peptidic bond between the Lys34 εNH2 and the α-COOH of the activated Lys. The ability of activated amino acids to make isopeptidic bonds with the εNH2 moiety of Lys lateral chains has already been demonstrated for S. cerevisiae AspRS [34]. Finally, YjeK could convert the EF-P bound α-lysine into β-lysine in presence of S-adenosyl methionine (SAM) and pyridoxal phosphate (PLP) (Fig 4A). The low activity of the E. coli YjeK on free (S)-α-lysine noted above could be due to the fact that the natural substrate is linked to EF-P [22]. The absence of the C-terminal dimerization domain in YjeK might allow it to access the EF-P-bound substrate.


Predicting the pathway involved in post-translational modification of elongation factor P in a subset of bacterial species.

Bailly M, de Crécy-Lagard V - Biol. Direct (2010)

Potential EF-P modification pathways. A- Mechanism in which YjeA acts first on free lysine (Lys) and attaches it to EF-P Lys34 which is then modified on EF-P into β-lysine by YjeK. B- Mechanism in which YjeK acts first to modify free lysine into β-lysine which is subsequently activated by YjeA and attached to EF-P lysine 34. EF-P N-terminal loop is indicated in yellow, Lys 34 is indicated in red, the modification appear on light brown and the AMP generated by YjeA during the activation of the Lys residue appear in blue. Potential and known substrates and cofactors of each enzyme are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821294&req=5

Figure 4: Potential EF-P modification pathways. A- Mechanism in which YjeA acts first on free lysine (Lys) and attaches it to EF-P Lys34 which is then modified on EF-P into β-lysine by YjeK. B- Mechanism in which YjeK acts first to modify free lysine into β-lysine which is subsequently activated by YjeA and attached to EF-P lysine 34. EF-P N-terminal loop is indicated in yellow, Lys 34 is indicated in red, the modification appear on light brown and the AMP generated by YjeA during the activation of the Lys residue appear in blue. Potential and known substrates and cofactors of each enzyme are indicated.
Mentions: In the first model (Fig. 4A), YjeA activates (S)-α-lysine into (S)-α-lysyl•AMP in presence of ATP and magnesium. This is coherent with the presence of AMP in the YjeA three dimensional structure (PDB: 3G1Z) and the conservation of the lysine binding residues in the YjeA active site (Fig. 3C). The activated (S)-α-lysine could then be transferred to the conserved Lys34 of EF-P to form an iso-peptidic bond between the Lys34 εNH2 and the α-COOH of the activated Lys. The ability of activated amino acids to make isopeptidic bonds with the εNH2 moiety of Lys lateral chains has already been demonstrated for S. cerevisiae AspRS [34]. Finally, YjeK could convert the EF-P bound α-lysine into β-lysine in presence of S-adenosyl methionine (SAM) and pyridoxal phosphate (PLP) (Fig 4A). The low activity of the E. coli YjeK on free (S)-α-lysine noted above could be due to the fact that the natural substrate is linked to EF-P [22]. The absence of the C-terminal dimerization domain in YjeK might allow it to access the EF-P-bound substrate.

Bottom Line: Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway.Zhulin and Mikhail Gelfand.For the full reviews, please go to the Reviewers' reports section.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA.

ABSTRACT

Background: The bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for protein synthesis. It is homologous to the eukaryotic translation initiation factor 5A (eIF5A). A highly conserved eIF5A lysine is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for eIF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a spermidine derivative in Escherichia coli. However, the biosynthesis pathway of this modification in the bacterial EF-P is yet to be elucidated.

Presentation of the hypothesis: Here we propose a potential mechanism for the post-translational modification of EF-P. By using comparative genomic methods based on physical clustering and phylogenetic pattern analysis, we identified two protein families of unknown function, encoded by yjeA and yjeK genes in E. coli, as candidates for this missing pathway. Based on the analysis of the structural and biochemical properties of both protein families, we propose two potential mechanisms for the modification of EF-P.

Testing the hypothesis: This hypothesis could be tested genetically by constructing a bacterial strain with a tagged efp gene. The tag would allow the purification of EF-P by affinity chromatography and the analysis of the purified protein by mass spectrometry. yjeA or yjeK could then be deleted in the efp tagged strain and the EF-P protein purified from each mutant analyzed by mass spectrometry for the presence or the absence of the modification. This hypothesis can also be tested by purifying the different components (YjeK, YjeA and EF-P) and reconstituting the pathway in vitro.

Implication of the hypothesis: The requirement for a fully modified EF-P for protein synthesis in certain bacteria implies the presence of specific post-translational modification mechanism in these organisms. All of the 725 bacterial genomes analyzed, possess an efp gene but only 200 (28%) possess both yjeA and yjeK genes. In the other organisms, EF-P may be modified by another pathway or the translation machinery must have adapted to the lack of EF-P modification. Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway.

Reviewers: This article was reviewed by Céline Brochier-Armanet, Igor B. Zhulin and Mikhail Gelfand. For the full reviews, please go to the Reviewers' reports section.

Show MeSH
Related in: MedlinePlus