Structure-activity relationships of different beta-lactam antibiotics against a soluble form of Enterococcus faecium PBP5, a type II bacterial transpeptidase

Abstract

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. beta-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel beta-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different beta-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Delta1-36 rPBP5). The soluble protein, Delta1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH(2)-terminal protein sequence was verified by amino acid sequencing. Using beta-lactams with different R1 side chains, we show that azlocillin has greater affinity for Delta1-36 rPBP5 than piperacillin and ampicillin (apparent K(i) = 7 +/- 0.3 microM, compared to 36 +/- 3 and 51 +/- 10 microM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k(2) = 15 +/- 4 M(-1) s(-1)). Meropenem demonstrates an affinity for Delta1-36 rPBP5 comparable to that of ampicillin (apparent K(i) = 51 +/- 15 microM) but is slower at acylating (apparent k(2) = 0.14 +/- 0.02 M(-1) s(-1)). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a beta-lactam, and suggests that the specific side chain interactions of beta-lactams could be modified to improve inactivation of resistant PBPs.

FIG. 1.

Ribbon representation of the structure of E. faecium Δ1-36 rPBP5.

FIG. 2.

Chemical structures of β-lactams used in these studies.

FIG. 3.

Determination of maximum F_I (F_{I max}) by reacting 200 μM BOCILLIN FL with Δ1-36 rPBP5 for 4 h.

FIG. 4.

Comparative binding studies (IC₅₀ determinations) performed with 200 μM BOCILLIN. Azlocillin demonstrated the greatest affinity for Δ1-36 rPBP5 of E. faecium.

FIG. 5.

Determination of second-order rate constants for competing β-lactams. Each β-lactam was competed with 500 μM BOCILLIN for 1 h at 37°C.

FIG. 6.

(Left) Experimentally observed conformation of the benzylpenicillinoyl moiety of the acyl-enzyme complex formed with Δ1-36 rPBP5. (Right) Modeled conformation of the penicillinoyl moiety of the acyl-enzyme complex formed between Δ1-36 rPBP5 and azlocillin, based on the benzylpenicillin complex. The carbonyl oxygen of the ureido side chain offers H-bonding interactions not present with other β-lactams.