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 (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 probable mode of association between actACP and actKR; we obtain and analyze a detailed model of the C7–C12-cyclized
octaketide within the actKR 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 (7R,12R) 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.