Fig. 3.

Identification of residues that contribute to KS–ACP specificity during chain elongation. (A) Schematic secondary structure of a typical ACP three-helix bundle. The approximate location (S) of the Ser residue onto which the phosphopantetheine arm is attached is shown. The secondary structure elements are mapped onto the tertiary structure in the ACP3 cartoon shown in B. Also shown is a multiple sequence alignment of ACP3, SHIV24, SHIV29, and ACP6 discussed in B. (B) An example of orthogonal KS–ACP recognition between modules 3 and 6 of DEBS. Also shown are two chimeric derivatives in which substitution of loop I of the ACP led to reversal of its preference for a KS partner. (C) Representative radio-thin-layer chromatography assay to quantify the relative preference of a given ACP for alternative KS partners. [KS3][AT3] and [KS6][AT6] refer to the ca. 190-kD homodimeric fragments of modules 3 and 6, respectively, that harbor both the KS and AT domains. (D) Site-directed mutagenesis of AYC79, a minimally altered chimeric derivative of ACP3 with greater preference for [KS6][AT6] than [KS3][AT3]. For the chain elongation activity assay, see SI Materials and Methods. Red, ACP3-derived sequences; green, ACP6-derived sequences. For steric reasons, an H26A mutation was necessary to obtain soluble protein (5).