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

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.

Figure 1

Genomic organisation of efp, yjeA and yjeK genes. A-Physical clustering of efp (in brown), yjeA (in pink) and yjeK (in orange) genes in several organisms. The black lines indicate that the genes are not contiguous in the genome. Examples of organisms and percentages for each genomic organisation among the 200 genomes that possess both yjeA and yjeK genes are indicated. The full list is given in Additional file 1. B and C- The sequence logos were created using an alignments of 9 amino acids from the EF-P protein sequences surrounding the position 34 generated in Clustal W2. The Logos were then generated by pasting the alignment in the WebLogo interface version 2.8.2 [39,40]. B- Logo for EF-P proteins from organisms that possess YjeA and YjeK (the list of sequences used is given in Additional file 3). C- Logo for EF-P proteins from organisms deprived of YjeA and YjeK.

Figure 2

Phylogenetic and structural analysis of the LAM family of proteins. A- Phylogenic tree generated with a subset YjeK and LAM proteins. Methods for alignment and tree construction are described in the text. This analysis shows that YjeK (in orange) and LAM (in blue) proteins forms distinct clades with relevant bootstrap values (923 for the LAM clade and 906 for the YjeK clade). The boxes correspond to the presence of the genes encoding for the protein indicated on top of the figure in the corresponding organism, white for genes present but not involved in a clustering, orange for genes that cluster with efp, and blue for genes that cluster with β-lysine acetyltransferase (Lysine degradation pathway). Accession numbers for the protein used can be found in Additional file 1. B- Three dimensional structure of LAM from Clostridium subterminale SB4 [24] (PDB: 2A5H) in blue with the C-terminal multimerization domain in pink, and 3D-model of YjeK from Acinetobacter baylyi based on C. subterminale SB4. The 3D model was build by using the homology method on the SWISS-MODEL web server [41-43].

Figure 3

Structural analysis of YjeA protein family. A- Three dimensional structure of YjeA from Salmonella typhimurium (PDB: 3G1Z) in pink and class II lysyl-tRNA synthetase (LysRS) from Escherichia coli [26] (PDB: 1BBU) in purple. The domains that constitute LysRS are indicated: catalytic core and anticodon binding domain (ABD). B- Merging of the two previous global structures and zoom into the active site and catalytic residues. The residues responsible for lysine binding are in pink for YjeA and in purple for the LysRS2. The lysine substrate present in LysRS2 structure is indicated in orange and the AMP present in YjeA structure is indicated in yellow. Red circles highlight the residues for which the 3 dimensional positions are not conserved.

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.

Figure 5

Wenn diagram. Red representing bacteria with efp; orange, a subset of those with the conserved. lysine; blue, bacteria with yjeA; and green, bacteria with yjeK (the colors, of course, are arbitrary, whereas the topology is not).