Purification, properties, and kinetics of enzymatic acylation with beta-lactams of soluble penicillin-binding protein 2a. A major factor in methicillin-resistant Staphylococcus aureus.

Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay

“…As shown in Fig. 4B, LY275858 (with an MIC for MRSA of 1 g/ml [11]) was a potent inhibitor of the nitrocefin-based enzymatic acylation activity of PBP 2aЈ. Methicillin and penicillin V, which are among the weakest ␤-lactams against PBP 2aЈ (11), were not inhibitory at 10 M. At a 1 mM concentration…”

“…When urea was gradually removed from the dissolved precipitate of LY275858-PBP 2aЈ by equilibrium dialysis, a soluble form of acylated PBP 2aЈ was produced. It appeared to be almost fully folded on the basis of the fluorescence spectrum and acylated, as indicated by the fluorescence-quenching phenomenon which is characteristic of ␤-lactam-acylated PBP 2aЈ (11). Nitrocefin-induced precipitation.…”

“…Purification and precipitation of PBP 2a. Purification of PBP 2aЈ was carried out by a previously published procedure (11). Escherichia coli cells expressing the modified mecA gene (in which the DNA sequence coding for the amino-terminal putative membrane-anchoring region of 21 residues was deleted) were grown in a fermentor, and PBP 2aЈ was purified from the soluble cell extract by ionexchange and affinity chromatographies.…”

“…The protein sample was excited at 295 nm (for preferential excitation of tryptophan residues), and the emission was measured in the 300-to 400-nm range. The enzymatic acylation activity of PBP 2aЈ under these conditions was monitored by the 125 I-labeled penicillin V acylation assay (11). To measure the extent of tertiary folding under partially denaturing conditions, the PBP 2aЈ-LY275858 precipitate was first dissolved in 8 M urea at a 1-mg/ml concentration and was subsequently diluted 10-fold in 1.5 to 3.0 M urea (final concentration) and incubated at room temperature for 1 h. Unacylated PBP 2aЈ was also treated under identical conditions.…”

“…Recently, using purified preparations of a soluble form of PBP 2a (PBP 2aЈ), we showed that the affinity parameters of PBP 2aЈ for various ␤-lactams vary in correlation with their respective MICs (2,11). The role of PBP 2a as the primary causative agent of ␤-lactam resistance was therefore established through in vitro biochemical studies.…”