Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli.
We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.
Takasuga
A
A
Department of Agricultural Chemistry, University of Tokyo.
Adachi
H
H
Ishino
F
F
Matsuhashi
M
M
Ohta
T
T
Matsuzawa
H
H
D000217:Acyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics* / Q000378:metabolism; D012694:Serine; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D012694:Serine; D000217:Acyltransferases; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D002918:Chymotrypsin; D014357:Trypsin