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Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.

Villet R, Fonvielle M, Busca P, Chemama M, Maillard AP, Hugonnet JE, Dubost L, Marie A, Josseaume N, Mesnage S, Mayer C, Valéry JM, Ethève-Quelquejeu M, Arthur M - Nucleic Acids Res. (2007)

Bottom Line: Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme.In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv).The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.

ABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

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Main steps of peptidoglycan synthesis in W. viridescens. The subunit consists of β-1-4-linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) substituted by a depsipeptide which is linked to the d-lactoyl group of MurNAc by an amide bond. The assembly of the subunit starts in the cytoplasm by the synthesis of UDP-MurNAc, the first precursor dedicated to peptidoglycan synthesis (37). In the following steps, the Mur ligases sequentially add l-Ala, d-Glu, l-Lys, attached to the γ carboxyl of d-Glu (d-iGlu) and the depsipeptide d-Ala-d-Lac to form the stem pentadepsipeptide l-Ala1-d-iGlu2-l-Lys3-d-Ala4-d-Lac5. The FemXWv aminoacyl transferase adds the first residue of the side chain onto this nucleotide precursor. Synthesis of the subunit proceeds by the transfer of the phospho-MurNAc-pentadepsipeptide moiety of UDP-MurNAc-pentadepsipeptide to the C55 lipid carrier undecaprenyl phosphate to form lipid intermediate I (undecaprenyl-PP-MurNAc-pentadepsipeptide or lipid I). The addition of GlcNAc to lipid I leads to lipid intermediate II [undecaprenyl-PP-MurNAc-(pentadepsipeptide)GlcNAc] or lipid II. The second and third residues of the l-Ala-l-Ser and l-Ala-l-Ser-l-Ala side chains are added to the lipid intermediates by unknown Fem transferases. The α carboxyl of d-iGlu2 is amidated in mature peptidoglycan (d-iGln2). The insets indicate the relative abundance of precursors and muropeptides, which was determined by the absorbance at 260 and 195 nm, respectively.
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Figure 1: Main steps of peptidoglycan synthesis in W. viridescens. The subunit consists of β-1-4-linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) substituted by a depsipeptide which is linked to the d-lactoyl group of MurNAc by an amide bond. The assembly of the subunit starts in the cytoplasm by the synthesis of UDP-MurNAc, the first precursor dedicated to peptidoglycan synthesis (37). In the following steps, the Mur ligases sequentially add l-Ala, d-Glu, l-Lys, attached to the γ carboxyl of d-Glu (d-iGlu) and the depsipeptide d-Ala-d-Lac to form the stem pentadepsipeptide l-Ala1-d-iGlu2-l-Lys3-d-Ala4-d-Lac5. The FemXWv aminoacyl transferase adds the first residue of the side chain onto this nucleotide precursor. Synthesis of the subunit proceeds by the transfer of the phospho-MurNAc-pentadepsipeptide moiety of UDP-MurNAc-pentadepsipeptide to the C55 lipid carrier undecaprenyl phosphate to form lipid intermediate I (undecaprenyl-PP-MurNAc-pentadepsipeptide or lipid I). The addition of GlcNAc to lipid I leads to lipid intermediate II [undecaprenyl-PP-MurNAc-(pentadepsipeptide)GlcNAc] or lipid II. The second and third residues of the l-Ala-l-Ser and l-Ala-l-Ser-l-Ala side chains are added to the lipid intermediates by unknown Fem transferases. The α carboxyl of d-iGlu2 is amidated in mature peptidoglycan (d-iGln2). The insets indicate the relative abundance of precursors and muropeptides, which was determined by the absorbance at 260 and 195 nm, respectively.

Mentions: The peptidoglycan is an essential component of the bacterial cell envelope, since it provides a mechanical barrier to the internal osmotic pressure of the cytoplasm (1). The peptidoglycan subunit is assembled by a series of cytoplasmic and membrane steps (Figure 1) that lead to formation of nucleotide (UDP-linked) and lipid intermediates. The complete subunit is translocated to the cell surface by an unknown mechanism and polymerized by the glycosyltransferases, that form the β-1-4 bonds linking the repeating GlcNAc–MurNAc units (2), and by the d,d-transpeptidases, that form peptide bonds between stem peptides in order to cross-link adjacent glycan strands (3). The latter activity is the target of β-lactam antibiotics that mimic the d-Ala4-d-Ala5 extremity of peptidoglycan precursors (4) and act as ‘suicide’ substrates in an essentially irreversible acylation reaction (5). In Gram-positive bacteria, β-lactam resistance is often due to so-called low-affinity penicillin-binding proteins (PBPs) that interact poorly with β-lactams and catalyze peptidoglycan cross-linking in the presence of the drug (3).Figure 1.


Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.

Villet R, Fonvielle M, Busca P, Chemama M, Maillard AP, Hugonnet JE, Dubost L, Marie A, Josseaume N, Mesnage S, Mayer C, Valéry JM, Ethève-Quelquejeu M, Arthur M - Nucleic Acids Res. (2007)

Main steps of peptidoglycan synthesis in W. viridescens. The subunit consists of β-1-4-linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) substituted by a depsipeptide which is linked to the d-lactoyl group of MurNAc by an amide bond. The assembly of the subunit starts in the cytoplasm by the synthesis of UDP-MurNAc, the first precursor dedicated to peptidoglycan synthesis (37). In the following steps, the Mur ligases sequentially add l-Ala, d-Glu, l-Lys, attached to the γ carboxyl of d-Glu (d-iGlu) and the depsipeptide d-Ala-d-Lac to form the stem pentadepsipeptide l-Ala1-d-iGlu2-l-Lys3-d-Ala4-d-Lac5. The FemXWv aminoacyl transferase adds the first residue of the side chain onto this nucleotide precursor. Synthesis of the subunit proceeds by the transfer of the phospho-MurNAc-pentadepsipeptide moiety of UDP-MurNAc-pentadepsipeptide to the C55 lipid carrier undecaprenyl phosphate to form lipid intermediate I (undecaprenyl-PP-MurNAc-pentadepsipeptide or lipid I). The addition of GlcNAc to lipid I leads to lipid intermediate II [undecaprenyl-PP-MurNAc-(pentadepsipeptide)GlcNAc] or lipid II. The second and third residues of the l-Ala-l-Ser and l-Ala-l-Ser-l-Ala side chains are added to the lipid intermediates by unknown Fem transferases. The α carboxyl of d-iGlu2 is amidated in mature peptidoglycan (d-iGln2). The insets indicate the relative abundance of precursors and muropeptides, which was determined by the absorbance at 260 and 195 nm, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175331&req=5

Figure 1: Main steps of peptidoglycan synthesis in W. viridescens. The subunit consists of β-1-4-linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) substituted by a depsipeptide which is linked to the d-lactoyl group of MurNAc by an amide bond. The assembly of the subunit starts in the cytoplasm by the synthesis of UDP-MurNAc, the first precursor dedicated to peptidoglycan synthesis (37). In the following steps, the Mur ligases sequentially add l-Ala, d-Glu, l-Lys, attached to the γ carboxyl of d-Glu (d-iGlu) and the depsipeptide d-Ala-d-Lac to form the stem pentadepsipeptide l-Ala1-d-iGlu2-l-Lys3-d-Ala4-d-Lac5. The FemXWv aminoacyl transferase adds the first residue of the side chain onto this nucleotide precursor. Synthesis of the subunit proceeds by the transfer of the phospho-MurNAc-pentadepsipeptide moiety of UDP-MurNAc-pentadepsipeptide to the C55 lipid carrier undecaprenyl phosphate to form lipid intermediate I (undecaprenyl-PP-MurNAc-pentadepsipeptide or lipid I). The addition of GlcNAc to lipid I leads to lipid intermediate II [undecaprenyl-PP-MurNAc-(pentadepsipeptide)GlcNAc] or lipid II. The second and third residues of the l-Ala-l-Ser and l-Ala-l-Ser-l-Ala side chains are added to the lipid intermediates by unknown Fem transferases. The α carboxyl of d-iGlu2 is amidated in mature peptidoglycan (d-iGln2). The insets indicate the relative abundance of precursors and muropeptides, which was determined by the absorbance at 260 and 195 nm, respectively.
Mentions: The peptidoglycan is an essential component of the bacterial cell envelope, since it provides a mechanical barrier to the internal osmotic pressure of the cytoplasm (1). The peptidoglycan subunit is assembled by a series of cytoplasmic and membrane steps (Figure 1) that lead to formation of nucleotide (UDP-linked) and lipid intermediates. The complete subunit is translocated to the cell surface by an unknown mechanism and polymerized by the glycosyltransferases, that form the β-1-4 bonds linking the repeating GlcNAc–MurNAc units (2), and by the d,d-transpeptidases, that form peptide bonds between stem peptides in order to cross-link adjacent glycan strands (3). The latter activity is the target of β-lactam antibiotics that mimic the d-Ala4-d-Ala5 extremity of peptidoglycan precursors (4) and act as ‘suicide’ substrates in an essentially irreversible acylation reaction (5). In Gram-positive bacteria, β-lactam resistance is often due to so-called low-affinity penicillin-binding proteins (PBPs) that interact poorly with β-lactams and catalyze peptidoglycan cross-linking in the presence of the drug (3).Figure 1.

Bottom Line: Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme.In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv).The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.

ABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

Show MeSH