Fig. 3. In vitro reconstitution of aminoacyl thioester formation and N-acylation by the CI-A-PCP tridomain from module 3 of IcoA. Deconvoluted mass spectra of IcoA module 3 CI-A-PCP tri-domain. (A) Resulting from incubation with ATP, l-Ser and (3R)-3-hydroxyoctanoyl (top), (3R)-3-hydroxydecanoyl (middle), or (3R)-3-hydroxyoctanoyl and (3R)-3-hydroxydecanoyl (bottom) pantetheine thioesters. (B) Following incubation with ATP, l-Ser and (3S)-3-hydroxyoctanoyl (top), or (3S)-3-hydroxydecanoyl (bottom) pantetheine thioesters. (C) Resulting from incubation of the H164A mutant with ATP, l-Ser and (3R)-3-hydroxyoctanoyl (top), or (3R)-3-hydroxydecanoyl (bottom) pantetheine thioesters. Dashed lines indicate the peaks corresponding to each condensed species, and the holo- and l-Ser-loaded species are labelled as indicated in the bottom right corner. Loading of l-Ser onto the PCP domain results in an 87 Da mass increase. For the wild type IcoA CI-A-PCP tri-domain, N-acylation of the serinyl-PCP thioester with the (3R)-3-hydroxyoctanoyl and (3R)-3-hydroxydecanoyl thioesters resulted in additional 142 and 170 Da mass increases, respectively. Analogous mass increases were observed when the (3S)-3-hydroxyoctanoyl and (3S)-3-hydroxydecanoyl thioesters were used, but the levels of product formation were lower, indicating that the CI domain prefers R-configured 3-hydroxyacyl thioesters. A small amount of product formation was observed when the H164A mutant of the IcoA CI-A-PCP tri-domain was used in place of the wild type protein, due to uncatalysed N-acylation of the serinyl thioester with the (3R)-3-hydroxyoctanoyl and (3R)-3-hydroxydecanoyl thioesters.
