Path to Actinorhodin: Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail.

In type II polyketide synthases (PKSs), which typically biosynthesize several antibiotic and antitumor compounds, the substrate is a growing polyketide chain, shuttled between individual PKS enzymes, while covalently tethered to an acyl carrier protein (ACP): this requires the ACP interacting with a series of different enzymes in succession. During biosynthesis of the antibiotic actinorhodin, produced by Streptomyces coelicolor , one such key binding event is between an ACP carrying a 16-carbon octaketide chain ( act ACP) and a ketoreductase ( act KR). Once the octaketide is bound inside act KR, it is likely cyclized between C7 and C12 and regioselective reduction of the ketone at C9 occurs: how these elegant chemical and conformational changes are controlled is not yet known. Here, we perform protein-protein docking, protein NMR, and extensive molecular dynamics simulations to reveal a probable mode of association between act ACP and act KR; we obtain and analyze a detailed model of the C7-C12-cyclized octaketide within the act KR active site; and we confirm this model through multiscale (QM/MM) reaction simulations of the key ketoreduction step. Molecular dynamics simulations show that the most thermodynamically stable cyclized octaketide isomer (7 R ,12 R ) also gives rise to the most reaction competent conformations for ketoreduction. Subsequent reaction simulations show that ketoreduction is stereoselective as well as regioselective, resulting in an S -alcohol. Our simulations further indicate several conserved residues that may be involved in selectivity of C7-12 cyclization and C9 ketoreduction. Detailed insights obtained on ACP-based substrate presentation in type II PKSs can help design ACP-ketoreductase systems with altered regio- or stereoselectivity.