Abstract
The domains of modular polyketide synthases (PKSs) collaborate to extend and process polyketide intermediates; however, most of their interactions with one another remain mysterious. We used AlphaFold 2 to investigate how acyl carrier proteins (ACPs) present intermediates to ketoreductases (KRs), processing domains capable of not only setting the stereochemical orientations of β-hydroxyl substituents but also of α-substituents. In modules that do not contain a dehydratase (DH), the A- and B-type KRs that, respectively, generate l- and d-oriented β-hydroxy groups are predicted to possess distinct ACP docking sites. In modules containing DHs, where A-type KRs are much less common, both KR types are predicted to possess an ACP-docking site equivalent to that of B-type KRs from modules without DHs. To investigate this most common ACP docking site, mutagenesis was performed on 20 residues of the KR from the second pikromycin module within the model triketide synthase P1-P2-P7. The least active variants are those with mutations to a conserved hydrophobe, 2 residues downstream of the LDD motif of B-type KRs, predicted to insert into a hole adjacent to the phosphopantetheinylated serine of ACP.