The 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 whilst 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 cœlicolor, one such key binding event is between an ACP carrying a 16-carbon octaketide chain (actACP) and a ketoreductase (actKR). Once the octaketide is bound inside actKR, 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 likely mode of association between actACP and actKR; we obtain and analyze a detailed model of the C7-C12-cyclized octaketide within actKR’s active site; and 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 (7S,12R) also gives rise to the most ‘reactive 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 cyclisation and C9 ketoreduction. The detailed insights obtained on ACP-based substrate presentation in type II PKSs will help with the design of nonendogenous ACP-ketoreductase systems capable of biosynthesizing non-natural polyketides.


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Supplementary material

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Input files, structures, parameters
Input files for protein-protein docking; structures of M1-M20, M10 14IB , M14 16IB and M17 1IA in PDB format; AMBER input (topologies and coordinates) for all runs in IA , I B , and II; modified force field parameters for the PPant moiety and octaketide portions of 2.
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Supporting Information (additional details)
Additional details on docking calculations, construction of starting structures, parametrization of isomer-conformers of 2, MD simulation and analysis, NMR data, details of QM/MM simulations, analysis of hydrogen bonds in MD runs in II.

Supplementary weblinks

Repository of Calculations
- Charge assignment calculations at the HF/6-31G(d) level - Thermodynamic stability of C7-C12 pentaketide fragments (B3LYP/6-31+G(d,p) and SCS-MP2)