Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by electrospray mass spectrometry
| Autor: | Y, Sun, M D, Bauer, W, Lu |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Staphylococcus aureus
Acylation Molecular Sequence Data Penicillins Muramoylpentapeptide Carboxypeptidase Mass Spectrometry Recombinant Proteins Bacterial Proteins Hexosyltransferases Peptidyl Transferases Serine Penicillin-Binding Proteins Indicators and Reagents Methicillin Resistance Amino Acid Sequence Carrier Proteins |
| Zdroj: | Journal of mass spectrometry : JMS. 33(10) |
| ISSN: | 1076-5174 |
| Popis: | Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by beta-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied beta-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the beta-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403. |
| Databáze: | OpenAIRE |
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