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

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains requ ire glycopeptides as inducers for synthesis of resistance proteins, which a llows 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 glycopeptid e-dependent Enterococcus faecium clinical isolates were determined. Each on e had a mutation consisting either in a 5-bp insertion at position 41 leadi ng to an early stop codon, an in-frame 6-bp deletion causing the loss of tw o residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P31 3 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular mo deling of the E. faecium enzyme, using the X-ray structure of the homologou s Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated resid ues were found either to interact directly with one of the substrates of th e enzymatic reaction (E13 and D295) or to stabilize the position of critica l residues in the active site. Maintenance of the 3-D structure in the vici nity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.