Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistantStaphylococcus aureus by electrospray mass spectrometry

✍ Scribed by Sun, Yiping; Bauer, Mark D.; Lu, Weiping

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 289 KB
• Volume: 33
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/(sici)1096-9888(1998100)33:10<1009::aid-jms717>3.0.co;2-4

✦ Synopsis

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the b-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by b-lactams is due to covalent modiÐcation of an active site serine residue. Based on the sequence alignment with well studied b-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S 403 XXK in PBP2a was tentatively identiÐed 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 identiÐcation of the active site serine in PBP2a is described. The covalent binding of the b-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 identiÐcation of the modiÐed 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.