Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics

Abstract

To understand the biochemical basis of resistance of bacteria to ␤-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k cat /K m values for the two purified enzymes (3,400 M ؊1 s ؊1 for S-PBP2x and 11.2 M ؊1 s ؊1 for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of ␤-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

Kinetics of β-Lactam Interactions with Penicillin-susceptible and -resistant Penicillin-binding Protein 2x Proteins from Streptococcus pneumoniae

“…Such digestion may have occurred during the E. coli expression of the recombinant protein or during the purification. Similar heterogeneous behavior of molecular mass has been reported for the same PBP2x protein (23). Upon incubation of the protein with pen-G, all three species increased their molecular mass by 335 Da as determined by ESMS, indicating that all of them were active in binding the ␤-lactam.…”

“…The value 1.5 ϫ 10 Ϫ5 s Ϫ1 determined for PBP2a of methicillin-resistant S. aureus indicates that the acyl-PBP2a adduct is stable with t1 ⁄2 Ͼ13 h. This k 3 value is also comparable with that of penicillinsensitive PBPs, indicating little role played by this step in the resistance mechanism of PBP2a (16). No detectable deacylation of the penicillin-Sp328 PBP2x R complex was observed upon incubation up to 90 min (indicating a k 3 value of Ͻ1 ϫ 10 Ϫ5 s Ϫ1 ) in a dilution/SDS-PAGE-based experiment (23). However, a value of 3 ϫ 10 Ϫ5 s Ϫ1 determined using an ESMS method was reported recently for the same PBP2x R complex (36).…”

“…Purification of the Recombinant PBP2x R to Homogeneity-A threestep purification protocol, different from the published procedures (17,23), was developed that offered the advantages of large scale, high yield, and efficiency. All operations were performed on a fast protein liquid chromatography purification system (Pharmacia) at 4°C.…”

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

“…In contrast, the flexible architecture of PBP2x from resistant strain SP328 results in an "open" active-site pocket that may be able to accommodate branched peptidoglycan precursors (27), for which it apparently exhibits a substrate preference, as will be discussed below. Although kinetic studies of low-affinity PBP2x also demonstrated that it has a reduced affinity for a structural analog of the linear stem peptide (69,117), studies with an analog of the branched stem peptide have not been done to confirm the substrate preference hypothesis.…”

TheponAGene ofEnterococcus faecalisJH2-2 Codes for a Low-Affinity Class A Penicillin-Binding Protein

“…Binding and/or inhibitory capacity of moenomycin and vancomycin antibiotics was determined. Acylation kinetics of the transpeptidase module of some of these PBPs with ␤-lactams were also analyzed (8,9,10,11,40,43).…”