Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by electrospray mass spectrometry.
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.
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Corporate Research Division, Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45253-8707, USA. suny.@pg.com
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D000215:Acylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D006602:Hexosyltransferases; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D016106:Methicillin Resistance / Q000502:physiology*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry*; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000737:chemistry; D010458:Peptidyl Transferases; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007202:Indicators and Reagents; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D012694:Serine; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase