Penicillin acylase from E. coli: unique gene-protein relation.

Area of Science:

• Molecular Biology
• Enzymology
• Protein Processing

Background:

• Penicillin G acylase (pac) is an enzyme crucial for antibiotic production.
• Understanding the genetic basis and processing of pac is vital for biotechnological applications.
• The E. coli pac gene encodes a precursor polypeptide requiring maturation.

Purpose of the Study:

• To determine the nucleotide sequence of the E. coli pac gene.
• To correlate the gene sequence with the primary structure of penicillin G acylase subunits.
• To elucidate the processing pathway of the pac gene product.

Main Methods:

• Nucleotide sequencing of the E. coli pac gene.
• Construction of plasmids for expressing truncated pac gene products.
• Analysis of protein accumulation and enzyme activity in E. coli.

Main Results:

• The pac gene open reading frame comprises four domains: signal peptide, alpha subunit, spacer peptide, and beta subunit.
• The two subunits are derived from a single precursor polypeptide, with the spacer peptide being removed during processing.
• Precursor lacking the signal sequence accumulates in the cytoplasm without processing or activity, indicating membrane translocation is essential.

Conclusions:

• Penicillin G acylase processing necessitates translocation across the cytoplasmic membrane.
• Processing follows a defined sequential pathway, and the spacer peptide is removed during this process.
• Cytoplasmic accumulation of the precursor polypeptide results in inactive enzyme.