Unexpected Inhibition of Peptidoglycan LD-Transpeptidase from Enterococcus faecium by the β-Lactam Imipenem

Abstract

peptide bond in the first step of the DD-transpeptidation reaction, leads to inactivation of the DD-transpeptidases. Because the acylenzymes typically have half-lives in the order of several hours, inactivation of the DD-transpeptidases by ␤-lactams is essentially irreversible in the scale of the generation time of bacteria. The DD-transpeptidases, which are highly redundant enzymes, are collectively the killing target of ␤-lactams, because peptidoglycan cross-linking is essential to the integrity of the cell wall. These enzymes belong to a large family of activesite serine acyl-transferases that bind penicillin covalently and are thus referred to as penicillin-binding proteins (PBPs) (4). The ␤-lactams are one of the oldest and still the most broadly used class of antibiotics for the treatment of severe infections despite the development of several resistance mechanisms, * This work was supported by the Fondation pour la Recherche Mé dicale (Equipe FRM 2006 (Grant DEQ200661107918)) and by NIAID, National Institutes of Health Grant R01 AI45626. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed.

Inactivation Kinetics of a New Target of β-Lactam Antibiotics

“…Bypass of the classical DD-transpeptidases by an LD-transpeptidase, as originally detected in an in vitro-selected E. faecium mutant, M512 (5), provides an alternate route of emergence of ␤-lactam resistance. In this mutant, Ldt fm is sufficient for peptidoglycan cross-linking because the chromosome of E. faecium harbors a single LD-transpeptidase gene, and 100% of the cross-links are generated by LD-transpeptidation in medium containing high concentrations of ampicillin (4). However, the classical DD-transpeptidation pathway remains functional, and, consequently, the selective pressure of imipenem applies on transpeptidases of DD and LD specificity.…”

“…However, this mutant was unable to grow in the presence of both ampicillin and imipenem. This indicates that the DD-transpeptidases retained an essential contribution to peptidoglycan cross-linking because ampicillin inactivates these enzymes but not Ldt fm (4). The selection procedure was therefore continued (10 M) was incubated with imipenem (150 M), and enzyme inactivation was monitored by spectrofluorimetry (gray curve).…”

“…For this approach, EI* was prepared by incubating enzyme (100 M) with imipenem (200 M). Excess imipenem was removed by size exclusion chromatography, and the purified acylenzyme was incubated for 1,000 min at room temperature and analyzed by mass spectrometry, as described previously (4). Under such conditions, no formation of native enzyme, imipenem, or hydrolyzed imipenem from the acylenzyme was detected.…”

“…The two modes of peptidoglycan cross-linking involve two stem peptides carried by adjacent glycan chains that act as acyl donor and acceptor. The DD-transpeptidases cleave the D-Ala 4 -D-Ala 5 bond at the extremity of a pentapeptide donor stem (hence the DD designation) and link the carbonyl of D-Ala 4 to the amine group located on the third residue of the acceptor (433 cross-links). The LD-transpeptidases use the same acceptor but act on a different peptide bond of the donor stem, which consists of a tetrapeptide in most bacterial species.…”

Peptidoglycan Hydrolases of Escherichia coli

“…Another possibility is that penems inhibit the LD-transpeptidases responsible for forming (L)-meso-A 2 pm3(D)-meso-A 2 pm cross-linkages and thereby contribute to the maintenance of an autolysin-sensitive PG structure. A penemsensitive LD-transpeptidase from Enterococcus faecium has been reported (160).…”