Topographical and functional investigation of Escherichia coli penicillin-binding protein 1b by alanine stretch scanning mutagenesis.
Penicillin-binding proteins (PBPs) are the targets of beta-lactam antibiotics. We have used a systematic five-alanine substitution method (called ASS [alanine stretch scanning] mutagenesis) to investigate the functional or structural role of various stretches of amino acids in the PBP1b of Escherichia coli. To probe the specific activity of each variant, the antibiotic discs assay was used with strain QCB1 (delta ponB) in the presence of cefaloridine, which totally inhibits the complementing action of PBP1a. This in vivo test has been combined with a quick and efficient in vitro test of the penicillin-binding activity of each of these variants with fluorescent penicillin. This approach has enabled us to show an unexpected role of the N-terminal and C-terminal tails of PBP1b. Moreover, we have established the correct position in PBP1b of the SMN motif that, with the SXXK and the KTG motifs, constitutes the signature of the penicilloyl serine transferases family. Finally, we have shown that the transglycosylase and the transpeptidase domains are separated by an inert linker region, where substitutions and insertions can be made without hindering the in vivo and in vitro activity of the protein.
Lefèvre
F
F
Institut de Pharmacologie et de Biologie Structurale, UPR 9062 du CNRS, Toulouse, France.
Rémy
M H
MH
Masson
J M
JM
D000409:Alanine / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002267:Muramoylpentapeptide Carboxypeptidase; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D002267:Muramoylpentapeptide Carboxypeptidase; D000409:Alanine