Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase

Abstract

Holo-acyl carrier protein synthase (ACPS) transfers the 4'- phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to Ser-36 of acyl carrier protein (ACP) in Escherichia coli. This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4'-PP. We have purified E. coli ACPS to near homogeneity by exploiting the ability to refold ACPS and reconstitute its activity after elution from an apo-ACP affinity column under denaturing conditions. N-terminal sequencing of ACPS allowed us to identify dpj, an essential gene of previously unknown function, as the structural gene for ACPS. We report herein the 70,000-fold purification of wild-type ACPS and the overproduction and initial characterization of recombinant ACPS from E. coli.