Structure-Activity Relationships of Different β-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. ␤-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 ␤-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 ␤-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (⌬1-36 rPBP5). The soluble protein, ⌬1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH 2 -terminal protein sequence was verified by amino acid sequencing. Using ␤-lactams with different R1 side chains, we show that azlocillin has greater affinity for ⌬1-36 rPBP5 than piperacillin and ampicillin (apparent K i ‫؍‬ 7 ؎ 0.3 M, compared to 36 ؎ 3 and 51 ؎ 10 M, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k 2 ‫؍‬ 15 ؎ 4 M ؊1 s ؊1 ). Meropenem demonstrates an affinity for ⌬1-36 rPBP5 comparable to that of ampicillin (apparent K i ‫؍‬ 51 ؎ 15 M) 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 ␤-lactam, and suggests that the specific side chain interactions of ␤-lactams could be modified to improve inactivation of resistant PBPs.Bacterial penicillin-binding proteins (PBPs) are members of the penicilloyl serine transferase family of enzymes (2,3,8,11). PBPs catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from the glycolipid precursor, lipid II (2). These reactions are composed of two parts: transglycosylation and transpeptidation. Transglycosylation joins the disaccharides of lipid II monomers to the peptidoglycan, creating the N-acetylmuramic acid and N-acetylglucosamine backbone of the murein sacculus. Transpeptidation cross-links the glycosidic backbone into a network of interlocking units that confers exceptional tensile strength. There are three broad types of PBPs (types I to III). Type I PBPs are multifunctional, multidomain proteins catalyzing both peptidoglycan polymerization and cross-linking. Unfortunately, penicillin resistance mediated by PBPs that are, or have become, insensitive to the inhibition by ␤-lactams is found in many clinically important pathogens. Some of the more problematic pathogens are Staphylococcus aureus (methicillinresistant S. aureus [MRSA]), coagulase-negative staphylococci (CNS), Neisseria gonorrhoeae, Streptococcus pneumoniae, Enterococcus faecium, and Enterococcus faecalis (6). The increasing prevalence and resistance of these pathogens are overcoming our best therapeutic efforts, since few antibiotics are available to treat these penicillin-resistant o...

Diversity of Penicillin-binding Proteins

“…However, considering that the amount of acyl-enzyme species formed is less than 10% of the total FmtA ⌬27 ⅐Bocillin complex (see below), we can calculate an approximate apparent dissociation constant of K d app Ϸ 60 M. This value is consistent with dissociation constants previously determined for ␤-lactam-resistant PBPs such as PBP2a of S. aureus (52), PBP5 of Enterococcus faecium (51), and PBP2x from Streptococcus pneumoniae (53).…”

“…The interaction of FmtA ⌬27 with Bocillin is characterized by a slow rate of formation of the acyl-enzyme species and a binding affinity that is in the range of ␤-lactam-resistant PBPs (51)(52)(53). Each of these observations indicates that FmtA is largely resistant to ␤-lactam inactivation.…”

Early Insights into the Interactions of Different β-Lactam Antibiotics and β-Lactamase Inhibitors against Soluble Forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3

“…Our methods were adapted from the work of Hujer et al (24) and Spratt (44). Unlike PBP assays conducted with purified membrane preparations, the PBPs in these assays are soluble and purified, such that host cell ␤-lactamases do not complicate the assay results (17,48).…”

History, Virulence Genes, Identification and Antimicrobial Resistance of Enterococcus faecalis Isolated from Diabetic Foot Patients

“…Enterococci have an intrinsic low vulnerability or resistance to β-lactams. Enterococcus faecalis naturally has least inhibitory concentricity (MICs) for penicillin of 2-8 mg L, these significant human microorganisms have been the topic of intense molecular revisions, together with Enterococcus hirae, which is more of a veterinary anxiety (Hujer et al, 2005).…”