Functional and Structural Analyses of the Split-Dehydratase Domain in the Biosynthesis of Macrolactam Polyketide Cremimycin

In the biosynthesis of the macrolactam antibiotic cremimycin, the 3-aminononanoic acid starter unit is formed via a non-2-enoyl acyl carrier protein thioester intermediate, which is presumed to be constructed by <i>cis</i>-acyltransferase (AT) polyketide synthases (PKSs) CmiP2, CmiP3, and CmiP4. While canonical <i>cis</i>-AT PKS modules are comprised of a single polypeptide, the PKS module formed by CmiP2 and CmiP3 is split within the dehydratase (DH) domain. Here, we report the enzymatic function and the structural features of this split-DH domain. <i>In vitro</i> analysis showed that the split-DH domain catalyzes the dehydration reaction of (<i>R</i>)-3-hydroxynonanoyl <i>N</i>-acetylcysteamine thioester (SNAC) to form (<i>E</i>)-non-2-enoyl-SNAC, suggesting that the split-DH domain is catalytically active in cremimycin biosynthesis. In addition, structural analysis revealed that the CmiP2 and CmiP3 subunits of the split-DH domain form a tightly associated heterodimer through several hydrogen bonding and hydrophobic interactions, which are similar to those of canonical DH domains of other <i>cis</i>-AT PKSs. These results indicate that the split-DH domain has the same function and structure as common <i>cis</i>-AT PKS DH domains.