Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft

Penicillin G acylase is a periplasmic protein, cytoplasmically expressed as a precursor polypeptide comprising a signal sequence, the A and B chains o f the mature enzyme (209 and 557 residues respectively) joined by a spacer peptide of 54 amino acid residues. The wild-type AB heterodimer is produced by proteolytic removal of this spacer in the periplasm. The first step in processing is believed to be autocatalytic hydrolysis of the peptide bond b etween the C-terminal residue of the spacer and the active-site serine resi due at the N terminus of the B chain. We have deter mined the crystal struc ture of a slowly processing precursor mutant (Thr263Gly) of penicillin G ac ylase from Escherichia coli, which reveals that the spacer peptide blocks t he entrance to the active-site cleft consistent with an autocatalytic mecha nism of maturation. In this mutant precursor there is, however, an unexpect ed cleavage at a site four residues from the active-site serine residue. An alyses of the stereochemistry of the 260-261 bond seen to be cleaved in thi s precursor structure and of the 263-264 peptide bond have suggested factor s that may govern the autocatalytic mechanism. (C) 2000 Academic Press.