Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site.
In order to determine the active site of penicillin-binding protein 3 of Escherichia coli (PBP3), the serine residue at position 307 was replaced with alanine, threonine or cysteine by oligonucleotide-directed site-specific mutagenesis. Since a unique BanII site exists at the position corresponding to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted in significant enrichment of the mutant plasmids. For mutagenesis, the gene coding for PBP3 (ftsI) was inserted into the expression cloning vector pIN-IIB. The hybrid protein produced was able to bind penicillin while mutant PBP3 in which serine-307 was replaced with either alanine or threonine did not lead to any detectable binding. However, contrary to the report of Broome-Smith et al. (1985) thiol-penicillin-binding protein 3, in which serine-307 was replaced with cysteine, was still able to bind penicillin. Replacement of serine-445 with an alanine residue had no effect on penicillin binding to PBP3.
D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D002267:Muramoylpentapeptide Carboxypeptidase; D009154:Mutation; D010400:Penicillin G / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D000217:Acyltransferases; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G