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

Abstract

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.

Type II Polyketide Synthases: A Bioinformatics‐Driven Approach

“… [16] Heterologous expression of the act minimal PKS together with TcmN, the first cyclase in tetracenomycin (tcm) pathway catalyzing the C9‐C14 cyclization, resulted in the selective production of UWM1 (Figure 4). [40] The biochemical in vitro assays, structural analyses, and molecular dynamics simulations suggested that ActIII likely first performs C7‐C12 cyclization and then reduces the C9 keto group of the ACP‐tethered polyketo intermediate [38b,f] …”

“…Hutchinson concluded that the minimal PKS, CYC, and KR components exhibit “context‐dependent” behavior based on the analyses of different cyclization outcomes obtained by various heterologous expression combinations using actinorhodin, daunorubicin, tetracenomycin, and jadomycin enzymes [62] . Indeed, this behavior could be explained by improper protein‐protein interaction among the enzymes, such as the reported KR‐ACP interaction between ActIII and the ActACP [38f,47b] . Elucidating the precise mechanism for the interaction between the minimal PKS, KR, CYC, and other enzymes during the polyketide cyclization remains to be a challenge for future studies of type II PKS enzymology.…”