Modeling Linear and Cyclic PKS Intermediates through Atom Replacement

Abstract

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs.

Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL

“…7 Type II carrier proteins, as opposed to type I, are standalone enzymes that must recognize up to five partner proteins in a particular order. When not interacting with a partner, the carrier protein has been shown to protect the extending substrate from reactive compounds in the complex cytosol.…”

“…Here the substrate localizes in a hydrophobic cleft between helix II and III of the acyl carrier protein (ACP). NMR structural studies of acylated E. coli fatty acid ACP reveals sequestration of chain lengths greater than C4, 7d-e which shields the growing metabolite from nucleophiles other than the cognate partner protein. 8 Similar interactions are also seen in type II PKS, where helix II and III of actinorhodin ACP were shown to interact with cyclic and linear intermediate analogs.…”

Polyketide mimetics yield structural and mechanistic insights into product template domain function in nonreducing polyketide synthases

“…2). Our current set of mimetics includes: a linear ring-opened 4c-mimetic 5a, and the corresponding linear isoxazole 4c-mimetic 5b; the bis-isoxazole 7c and 8c-mimetics 5c and 5d (10). In addition, to probe cyclization specificity, we also prepared the cyclized and aromatized polyketide mimics 5e and 5f (10) (Fig.…”

“…Second, the high reactivity of the native 20-carbon poly-β-keto intermediate 1 prevents its de novo synthesis. Recently, a general strategy for the design and synthesis of stable mimics was developed using the concept of "atom replacement" (10). Conceptually, isoxazoles linked by thioethers are visualized to parallel the alternating pattern of methylene and carbonyl groups of NR-PKS intermediates.…”

“…Conceptually, isoxazoles linked by thioethers are visualized to parallel the alternating pattern of methylene and carbonyl groups of NR-PKS intermediates. Furthermore, the native thioester within these substrates was replaced by an isosteric amide bond to further stabilize the pantetheine-substrate connection (10,11). In this study, we prepared 4′-phosphorylated and unphosphorylated atom-replaced mimetics selected to represent substrate chain lengths of 8-16 carbon units of the corresponding linear and proposed monocyclic intermediates.…”