Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases

Abstract

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

Structural studies suggest aggregation as one of the modes of action for teixobactin

“…This was not possible in the case of lipid II titrations because the resonances for the bound form of teixobactin were broadened beyond detection due to aggregation. Instead, we fitted intensities of the peaks from free teixobactin as a function of lipid II concentration to extract apparent association constants26 ( K a is the inverse of K d ; see eqn (1) in methods, see Fig. ESI8–11, Tables ESI4, 5†).…”

Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

“…In light of the absence of conventional SIMs, we used a recently developed chemical biology approach, known as carbene footprinting [105][106][107] , to comprehensively map SUMO2 interactions along XRCC4. In contrast to other high-resolution PPI methods, this technology requires little starting material and is unrestricted by the molecular weight of the targeted protein as well as the affinity of the interaction.…”

“…Reduced peptide labelling in the presence of a binding partner, indicate the residues of the peptide as potential binding sites due to surface masking. In addition, unmasking of peptides labellings can occur, and both masking and unmasking can indicate interaction-induced conformational changes or rearrangements leading to a change in exposure of residues to solvent, and therefore labelling [105][106][107] (Fig. 4A).…”

A 3‐hydroxy‐3‐methylglutaryl‐CoA synthase‐based probe for the discovery of the acyltransferase‐less type I polyketide synthases

“…The biosynthetic potential of polyketides in environmental samples is typically surveyed using conserved KS domains in type I or type II PKSs (Savic & Vasiljevic, 2006). One limitation is that a single bacterial strain in a typical soil sample may contain dozens of KS domains, due to the presence of several PKS BGCs and multiple PKS modules in each type I PKS (Jenke-Kodama et al, 2005;Jenner et al, 2018;Khosla et al, 2014). Therefore, the accurate identification of KSs in soil samples would rely on the deep sequencing of environmental DNAs, to reduce the false-negative results (Crits-Christoph et al, 2018).…”