Identification, purification, and characterization of transpeptidase and glycosyltransferase domains of Streptococcus pneumoniae penicillin-binding protein 1a.
Resistance to beta-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both beta-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.
Di Guilmi
A M
AM
Institut de Biologie Structurale Jean-Pierre Ebel (CEA/CNRS), 38027 Grenoble Cedex 1, France.
Mouz
N
N
Andrieu
J P
JP
Hoskins
J
J
Jaskunas
S R
SR
Gagnon
J
J
Dideberg
O
O
Vernet
T
T
D001426:Bacterial Proteins; D002352:Carrier Proteins; D010450:Endopeptidases / Q000378:metabolism; D005982:Glutathione Transferase; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments; D010449:Peptide Mapping; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D014357:Trypsin / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010458:Peptidyl Transferases; D016695:Glycosyltransferases; D006602:Hexosyltransferases; D005982:Glutathione Transferase; D010450:Endopeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D014357:Trypsin