title: Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated.
abstract: Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.
authors.affiliation: Institut de Biologie Structurale Jean-Pierre Ebel (CEA/CNRS), 38027 Grenoble Cedex 1, France.
authors.forename: A M
authors.initials: AM
authors.lastname: di Guilmi
authors.forename: N
authors.initials: N
authors.lastname: Mouz
authors.forename: L
authors.initials: L
authors.lastname: Martin
authors.forename: J
authors.initials: J
authors.lastname: Hoskins
authors.forename: S R
authors.initials: SR
authors.lastname: Jaskunas
authors.forename: O
authors.initials: O
authors.lastname: Dideberg
authors.forename: T
authors.initials: T
authors.lastname: Vernet
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002439:Cefotaxime / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D016764:Cell Polarity; D004352:Drug Resistance, Microbial; D016695:Glycosyltransferases / Q000235:genetics / Q000302:isolation & purification*; D006602:Hexosyltransferases; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments / Q000302:isolation & purification; D010458:Peptidyl Transferases; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D008055:Lipids; D008565:Membrane Proteins; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010458:Peptidyl Transferases; D016695:Glycosyltransferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D014357:Trypsin; D002439:Cefotaxime