title: 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.
authors.affiliation: Louis Stokes Cleveland Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106, USA.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hujer
authors.forename: Malgosia
authors.initials: M
authors.lastname: Kania
authors.forename: Thomas
authors.initials: T
authors.lastname: Gerken
authors.forename: Vernon E
authors.initials: VE
authors.lastname: Anderson
authors.forename: John D
authors.initials: JD
authors.lastname: Buynak
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Ge
authors.forename: Patrick
authors.initials: P
authors.lastname: Caspers
authors.forename: Malcolm G P
authors.initials: MG
authors.lastname: Page
authors.forename: Louis B
authors.initials: LB
authors.lastname: Rice
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000215:Acylation; D000465:Algorithms; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001390:Azlocillin / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D015780:Carbapenems / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D016984:Enterococcus faecium / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D000077731:Meropenem; D008958:Models, Molecular; D010400:Penicillin G / Q000494:pharmacology; D046915:Penicillin-Binding Proteins / Q000037:antagonists & inhibitors*; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors*; D010957:Plasmids; D013329:Structure-Activity Relationship; D013845:Thienamycins / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D046915:Penicillin-Binding Proteins; D013845:Thienamycins; D010458:Peptidyl Transferases; D000077731:Meropenem; D001390:Azlocillin; D010400:Penicillin G