Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105.
The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the beta-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the alpha subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.
Sizmann
D
D
Lehrstuhl für Mikrobiologie, Universität München, Federal Republic of Germany.
Keilmann
C
C
Böck
A
A
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004755:Enterobacteriaceae / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial / Q000502:physiology*; D006090:Gram-Negative Bacteria / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010957:Plasmids / Q000502:physiology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000502:physiology; D021382:Protein Sorting Signals / Q000378:metabolism; D011549:Pseudomonas / Q000235:genetics
D010455:Peptides; D021382:Protein Sorting Signals; D000581:Amidohydrolases; D010399:Penicillin Amidase