Genetic analyses of processing involving C-terminal cleavage in penicillin-binding protein 3 of Escherichia coli.
The processing of Escherichia coli penicillin-binding protein 3 (PBP 3) was investigated by gene manipulation for producing hybrid and truncated PBP 3 molecules. The hybrid PBP 3 was processed when the N-terminal 40 residues of PBP 3 were replaced by the murein lipoprotein signal peptide which lacked the cysteine residue for processing and followed by seven extra linker residues. In contrast, the PBP 3 molecules truncated at Thr-560 (28-residue deletion) or at Thr-497 (91-residue deletion) were not processed, and those truncated at Phe-576 (12-residue deletion) were processed at a greatly reduced rate. The results indicate that the C-terminal part, rather than the N-terminal part, is involved in the processing. This was supported by the result that the purified mature PBP 3 retained the complete N-terminal sequence with Met for translation initiation. The cleavage at the C-terminal region was shown by the loss of [35S]cysteine label when the cysteine-free hybrid PBP 3 joined to a cysteine-rich extra peptide tail was processed into the mature form. Confirmative assays for processing of PBP 3 were aided by a newly found prc mutant, defective in the processing involving the C-terminal region. A plasmid that directs PBP 3 truncated at Thr-560 complemented a thermosensitive PBP 3 mutation, but the truncated product was unstable in vivo. This suggests the importance of C-terminal hydrophobic regions that terminate at Leu-558 to PBP 3 functioning and the requirement of further-distal peptides for the stability of PBP 3.
Hara
H
H
National Institute of Genetics, Shizuoka-ken, Japan.
Nishimura
Y
Y
Kato
J
J
Suzuki
H
H
Nagasawa
H
H
Suzuki
A
A
Hirota
Y
Y
D000217:Acyltransferases / Q000235:genetics*; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002872:Chromosome Deletion; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005838:Genotype; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics*; D002267:Muramoylpentapeptide Carboxypeptidase; D009154:Mutation; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011499:Protein Processing, Post-Translational
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; D003545:Cysteine