Changing glycine 21 for glutamic acid in the beta-subunit of penicillin G acylase from Kluyvera citrophila prevents protein maturation.

Abstract

Active penicillin acylase from Kluyvera citrophila strain ATCC 21,285 consists of two different alpha- and beta-subunits derived from a single precursor by post-translational processing. Using the chemical mutagen hydroxylamine we have treated plasmid pYKD59 containing the active penicillin acylase gene (pga) from K. citrophila and have generated different point mutant penicillin acylase genes, one producing a maturation deficient precursor. This point mutation has changed the glycine 310 residue of the precursor for a glutamic acid (residue number 21 of the mature beta-subunit). The introduction of a charged residue in this position did not prevent translocation of the precursor to the periplasm but the resultant molecule was not able to undergo subsequent post-translational modification to yield the active protein.