Document Detail


Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by electrospray mass spectrometry.
MedLine Citation:
PMID:  9821332     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Y Sun; M D Bauer; W Lu
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of mass spectrometry : JMS     Volume:  33     ISSN:  1076-5174     ISO Abbreviation:  J Mass Spectrom     Publication Date:  1998 Oct 
Date Detail:
Created Date:  1998-12-22     Completed Date:  1998-12-22     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9504818     Medline TA:  J Mass Spectrom     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1009-16     Citation Subset:  IM    
Affiliation:
Corporate Research Division, Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45253-8707, USA. suny.@pg.com
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MeSH Terms
Descriptor/Qualifier:
Acylation
Amino Acid Sequence
Bacterial Proteins*
Carrier Proteins / chemistry*
Hexosyltransferases*
Indicators and Reagents
Mass Spectrometry
Methicillin Resistance / physiology*
Molecular Sequence Data
Muramoylpentapeptide Carboxypeptidase / chemistry*
Penicillin-Binding Proteins
Penicillins / chemistry
Peptidyl Transferases*
Recombinant Proteins / chemistry
Serine / chemistry*
Staphylococcus aureus / chemistry,  metabolism*
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Carrier Proteins; 0/Indicators and Reagents; 0/Penicillin-Binding Proteins; 0/Penicillins; 0/Recombinant Proteins; 56-45-1/Serine; EC 2.3.2.12/Peptidyl Transferases; EC 2.4.1.-/Hexosyltransferases; EC 3.4.17.8/Muramoylpentapeptide Carboxypeptidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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