Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold

Abstract

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.

Insights into modular polyketide synthase loops aided by repetitive sequences

“…To determine the lengths of these loops, their interfaces with structured elements were identified. Absences of optional features such as DH η1, DH α4 (the terminal helix observed in a cremimycin DH, PDB 6K97), and ACP α1 were considered 23‐25 . Which residues are unstructured in the loop between KR and ACP in β‐modules is apparent; however, which residues are unstructured in the loop between these domains in γ‐ and δ‐modules is less clear.…”

“…ACP (n = 949): No insertions were observed within this 100‐residue, helical domain. This analysis includes α1 (often referred to as “helix 0”), which is rarely absent 25 …”

Insights into Modular Polyketide Synthase Loops Aided by Repetitive Sequences

“…To determine the length of flexible interdomain loops, their interfaces with structured elements were identified. Absences of optional features such as DH η1, DH α4 (the terminal helix observed in a cremimycin DH, PDB 6K97), and ACP α1 were considered [24][25][26]. Which residues are unstructured in the loop between KR and ACP in β-modules is apparent; however, which residues are unstructured in the loop between these domains in γ-and δ-modules is less clear.…”

“…ACP [n=949]: No insertions were observed within this 100-residue, helical domain. This analysis includes α1 (often referred to as "helix 0"), which is rarely absent [26]. DDs [n=388]: Most of the docking domain motifs, C DD and N DD, could be grouped into Class 1a (n=226), Class 1b (n=63), or Class 2 (n=70) (Supplementary Figures 9-11) [7].…”

Discovery of natural products from bacilli and pseudomonas for biocontrol of plant diseases

“…KS domain which catalyzes C-C bond formation (Broadhurst et al, 2003). The AT domain recruits a building block (usually methylmalonyl-CoA) and transfers its α-carboxyacyl moiety through a thioester linkage to the 4ʹphosphopantetheine (Ppant) arm of an ACP domain (Moretto et al, 2019). The ACP domain is responsible for tethering and shuttling the extender unit or the polyketide intermediate (Hwang et al, 2020).…”