Abstract
The biosynthesis of polyketides by type I modular polyketide synthases (PKS) relies on co-ordinated interactions between acyl carrier protein (ACP) domains and catalytic domains within the megasynthase. Despite the importance of these interactions, and their implications for biosynthetic engineering efforts, they remain poorly understood. Here, we report the molecular details of the interaction interface between an ACP domain and a ketoreductase (KR) domain from a trans-acyltransferase (trans-AT) PKS. Using a high-throughput mass spectrometry (MS)-based assay in combination with scanning alanine mutagenesis, residues contributing to the KR-binding epitope of the ACP domain were identified. Application of carbene footprinting revealed the ACP-binding site on the KR domain surface, and molecular docking simulations driven by experimental data allowed production of an accurate model of the complex. Interactions between ACP and KR domains from trans-AT PKSs were found to be specific for their cognate partner, indicating highly optimised interaction interfaces driven by evolutionary processes. Using detailed knowledge of the ACP:KR interaction epitope, an ACP domain was engineered to interact with a non-cognate KR domain partner. The results provide novel, high resolution insights into the ACP:KR interface and offer valuable rules for future engineering efforts of biosynthetic assembly lines.
Highlights
Polyketides constitute a valuable family of natural products, many of which nd application in both medicine and agriculture.[1,2] Despite extraordinary structural complexity and diversity, the chemical logic underpinning their biosynthesis is elegantly simple; the head-to-tail decarboxylative condensation of malonyl and acyl units to generate linear carbon frameworks, upon which additional structural diversi cation can be applied.[3]
Initial experiments revealed that PksJ KR2 was able to catalyse complete conversion of 3-keto-butyryl-PksJ ACP4 to (3S)-3-hydroxy-butyryl-PksJ ACP4 in the presence of NADPH a er a 10 min incubation period
We have elucidated molecular details of the interaction interface between an acyl carrier protein (ACP) domain and a KR domain from a trans-acyl transferase (AT) polyketide synthases (PKS), allowing production of a docked model driven by residue-level experimental data
Summary
Polyketides constitute a valuable family of natural products, many of which nd application in both medicine and agriculture.[1,2] Despite extraordinary structural complexity and diversity, the chemical logic underpinning their biosynthesis is elegantly simple; the head-to-tail decarboxylative condensation of malonyl and acyl units to generate linear carbon frameworks, upon which additional structural diversi cation can be applied.[3]. We apply a highly complementary set of techniques to elucidate residue-level details underpinning the interaction interface between a cognate pair of ACP (PksJ ACP4) and KR (PksJ KR2) domains from module 3 of the bacillaene trans-AT PKS (Fig. 1A).
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