Modular polyketide synthases (PKSs) catalyze the biosynthesis of medicinally important natural products through an assembly-line mechanism. Consequently, PKSs have been the focus of protein engineering efforts aimed at harnessing their programmable biochemistry. Several studies have established the central role played by protein-protein interactions involving both linker and domain regions in this process.

The interface between ketosynthase (KS) and acyl carrier protein (ACP) domains is involved in two settings: chain transfer from an upstream donor ACP to downstream acceptor KS domain (ACP_n-1 – KS_n), and chain elongation reaction catalyzed by ACP_n and KS_n (ACP_n – KS_n). Studies have shown that ACP domains are not functionally interchangeable in either case. Indeed, mismatched ACP-KS pairs have severely compromised kinetic parameters and in the extreme those exhibiting orthogonal specificities have been identified. Consequently, our ability to construct functional PKS chimeras is dependent on a clear understanding of the mechanism of intra and inter modular communication between the various enzyme partners.

A strategy employing fusion protein construction and site-directed mutagenesis was utilized to map the ACP domain residues involved in mediating ACP_n – KS_n specificity. The results strongly suggested that structural elements distinct from the so called “recognition helix” (helix II) play a critical role in this process. The same methodology was applied to elucidate the interaction interface at the ACP_n-1 – KS_n junction. To aid visualization of these abovementioned interfaces, docking models were generated utilizing experimental constraints. Together, these experimental results and models will facilitate future efforts directed towards rational engineering of hybrid PKSs.