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

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. P-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 P-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 NH2-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 muM, compared to 36 +/- 3 and 51 +/- 10 muM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k(2) = 15 +/- 4M(-1) s(-1)). Meropenem demonstrates an affinity for Delta1-36 rPBP5 comparable to that of ampicillin (apparent K-2 = 51 Delta 15 muM) 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 P-lactam, and suggests that the specific side chain interactions of beta-lactams could be modified to improve inactivation of resistant PBPs.