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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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Related in: MedlinePlus

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].
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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].

Mentions: yjeK encodes a homologue of lysine 2,3 aminomutase (LAM) involved in lysine catabolism [22]. However, it was shown in vitro that E. coli YjeK catalyzes the conversion of (S)-α-lysine to (R)-β-lysine and not of (S)-α-lysine to (S)-β-lysine like classical LAM enzymes [22]. The E. coli YjeK catalytic efficiency is quite low compared to the LAM catalyzed reaction (0.1% of the activity of the Clostridium subterminale SB4 LAM [22]). (S)-α-lysine might not therefore be the real in vivo substrate of the YjeK enzyme. Primary sequence alignment analysis on 95 LAM/YjeK homologs were performed using Clustal W2 [23] and revealed that the LAM (YjeK) that clusters with the efp gene can be separated from the canonical LAM involved in Lys degradation pathway. The major difference between the two families is that the YjeK proteins lack the C-terminal multimerization domain present in the LAM family of proteins [24] (Fig. 2B). Further phylogenetic analysis on 24 LAM/YjeK homologs was performed on 118 amino acid sequences present in the N-terminus active site and conserved between the YjeK and LAM subfamilies (Fig. 2A). The amino acid sequences were aligned using the ClustalW2 algorithm with default parameters [23]. Phylogenetic analyses were carried out by employing the Phylip 3.68 program package [25]. Distance-based matrices were generated between all pairs of sequences using the Jones-Taylor-Thornton matrix as employed in Protdist (Phylip). Phylogenetic trees were generated from these matrices using the neighbour-joining method as implemented in Neighbor (Phylip). Reliability of branches was determined with the bootstrap method of 1000 replicates using Seqboot (Phylip). The final tree was generated with Consense (Phylip). This analysis showed that despite the fact that YjeK from E. coli is 33% identical to LAM from C. subterminale SB4, the two enzyme subfamilies form two distinct clades on the YjeK/LAM phylogenetic tree separated by bootstrap scores ranging from 923 to 906 (Fig. 2A). Genome neighbourhood analyses were also used to split the two families. We observed that ablA (LAM) genes physically cluster mainly with other lysine degradation gene such as ablB (β-lysine acetyltransferase) in several organisms (See Fig. 2A and Additional file 1). On the contrary, yjeK genes cluster mainly with efp homologs but never with ablB genes (See Fig. 2A and Additional file 1). In genomes such as Syntrophus aciditrophicus and Desulfuromonas acetoxidans, where both yjeK and ablA genes are present, one was found to cluster with efp and the other with ablB respectively (Fig. 2A). The combination of structural, phylogenetic and physical clustering pattern differences between YjeK and LAM enzymes were used to split the LAM family of proteins into two subfamilies that will be referred to as LAM and YjeK from hereon and suggest that these families have distinct functions, the first in lysine catabolism, the second related to EF-P.


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)

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].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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].
Mentions: yjeK encodes a homologue of lysine 2,3 aminomutase (LAM) involved in lysine catabolism [22]. However, it was shown in vitro that E. coli YjeK catalyzes the conversion of (S)-α-lysine to (R)-β-lysine and not of (S)-α-lysine to (S)-β-lysine like classical LAM enzymes [22]. The E. coli YjeK catalytic efficiency is quite low compared to the LAM catalyzed reaction (0.1% of the activity of the Clostridium subterminale SB4 LAM [22]). (S)-α-lysine might not therefore be the real in vivo substrate of the YjeK enzyme. Primary sequence alignment analysis on 95 LAM/YjeK homologs were performed using Clustal W2 [23] and revealed that the LAM (YjeK) that clusters with the efp gene can be separated from the canonical LAM involved in Lys degradation pathway. The major difference between the two families is that the YjeK proteins lack the C-terminal multimerization domain present in the LAM family of proteins [24] (Fig. 2B). Further phylogenetic analysis on 24 LAM/YjeK homologs was performed on 118 amino acid sequences present in the N-terminus active site and conserved between the YjeK and LAM subfamilies (Fig. 2A). The amino acid sequences were aligned using the ClustalW2 algorithm with default parameters [23]. Phylogenetic analyses were carried out by employing the Phylip 3.68 program package [25]. Distance-based matrices were generated between all pairs of sequences using the Jones-Taylor-Thornton matrix as employed in Protdist (Phylip). Phylogenetic trees were generated from these matrices using the neighbour-joining method as implemented in Neighbor (Phylip). Reliability of branches was determined with the bootstrap method of 1000 replicates using Seqboot (Phylip). The final tree was generated with Consense (Phylip). This analysis showed that despite the fact that YjeK from E. coli is 33% identical to LAM from C. subterminale SB4, the two enzyme subfamilies form two distinct clades on the YjeK/LAM phylogenetic tree separated by bootstrap scores ranging from 923 to 906 (Fig. 2A). Genome neighbourhood analyses were also used to split the two families. We observed that ablA (LAM) genes physically cluster mainly with other lysine degradation gene such as ablB (β-lysine acetyltransferase) in several organisms (See Fig. 2A and Additional file 1). On the contrary, yjeK genes cluster mainly with efp homologs but never with ablB genes (See Fig. 2A and Additional file 1). In genomes such as Syntrophus aciditrophicus and Desulfuromonas acetoxidans, where both yjeK and ablA genes are present, one was found to cluster with efp and the other with ablB respectively (Fig. 2A). The combination of structural, phylogenetic and physical clustering pattern differences between YjeK and LAM enzymes were used to split the LAM family of proteins into two subfamilies that will be referred to as LAM and YjeK from hereon and suggest that these families have distinct functions, the first in lysine catabolism, the second related to EF-P.

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