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Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium

¹ Unité des Agents Antibactériens, Institut Pasteur, Paris, France
² Centre d'Ingénierie Biomoléculaire, Université Libre de Bruxelles, Brussels, Belgium
³ Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, Brussels, Belgium

Reprint requests to: Dr. Patrice Courvalin, Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Dr Roux 75724 Paris CEDEX 15, France; e-mail: pcourval{at}pasteur.fr; fax: 33-1-45-68-83-19.

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 G, G99 R, V241 D, D295 G, P313 L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.

Keywords: D-Ala:D-Ala ligase; glycopeptide-dependence; DNA sequencing; comparative molecular modeling

Abbreviations: D-Ala, D-Alanine • D-Ala:D-Ala ligase, D-Alanine:D-Alanine ligase • 3-D, three-dimensional

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