Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay.
We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.
Roychoudhury
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Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46285, USA.
Kaiser
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Brems
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Yeh
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D000215:Acylation; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002511:Cephalosporins / Q000494:pharmacology; D002863:Chromogenic Compounds / Q000737:chemistry; D006602:Hexosyltransferases; D007700:Kinetics; D007769:Lactams; D013058:Mass Spectrometry; D016106:Methicillin Resistance / Q000502:physiology*; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000378:metabolism*; D010404:Penicillin V / Q000494:pharmacology; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000494:pharmacology; D010458:Peptidyl Transferases; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D014508:Urea / Q000737:chemistry; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002511:Cephalosporins; D002863:Chromogenic Compounds; C103495:LY 275858; D007769:Lactams; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D013844:Thiazoles; D047090:beta-Lactams; D014508:Urea; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; C021720:nitrocefin; D010404:Penicillin V