pubmed.ncbi.nlm.nih.gov

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

  • ️Tue Jan 01 2019

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

Luisa Moretto et al. Sci Rep. 2019.

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Module organization for the three subunits of the mycolactone PKS system (MLSA1, MLSA2 and MLSB). The structure of mycolactone is colour coded to match the subunits responsible for synthesizing each segment. mH0ACPa and mH0ACPb are shaded yellow and orange, respectively. A1- and B1-type KR domains are white and magenta, respectively. DH domains that are predicted to be inactive are marked with diagonal black lines. Domain abbreviations: KS, ketosynthase; AT, acyltransferase; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; CP, acyl carrier protein; TE, thioesterase.

Figure 2
Figure 2

(A) Backbone overlay of ribbon representations for the final ensemble of structures for apo mH0ACPa, coloured from blue at the N-terminus to red at the C-terminus; and (B) cartoon representation of the lowest energy structure of mH0ACPa, with Ser11141 shown in red spheres. (C) Backbone overlay of ribbon representations for the final ensemble of structures for apo mH0ACPb, coloured from blue at the N-terminus to red at the C-terminus; and (D) cartoon representation of the lowest energy structure of mH0ACPb, with Ser13830 shown in red spheres.

Figure 3
Figure 3

Superposition of ribbon representations of lowest energy solution structures for (A) apo mH0ACPa (red) and apo mH0ACPb (yellow); and (B) apo mACPb (magenta; PDB-ID 5HVC) and apo mH0ACPb (yellow). (C) Superposition of ensembles for apo mH0ACPa (red) and apo dACP2 (blue; PDB-ID 2JU1).

Figure 4
Figure 4

Average 1HN/15N chemical shift differences (Δδav) plotted as a function of residue number, between: (A) apo mACPb and apo mH0ACPb; (B) holo mH0ACPb and apo mH0ACPb; (C) holo mH0ACPa and apo mH0ACPa; and (D) holo mH0ACPa and β-hydroxybutyryl-mH0ACPa. Schematics defining the boundaries of secondary structure elements in apo mH0ACPb and apo mH0ACPa are shown above panels (A) and (C), respectively, with asterisks indicating where the prosthetic groups are attached.

Figure 5
Figure 5

Underneath a schematic defining the boundaries of the secondary structure elements in apo mH0ACPa, nuclear spin relaxation parameters for backbone amide sites are plotted as a function of residue number for: (A) the 15N longitudinal relaxation rate, R1; (B) the 15N transverse relaxation rate, R2; (C) the {1H}-15N nuclear Overhauser effect ratio (I’/I0, where I’ is the intensity when the 1H spectrum has been saturated and I0 is the intensity in the reference spectrum); and (D) the Lipari-Szabo the order parameter, S2.

Figure 6
Figure 6

Representative ITC thermograms (upper panels) and isotherm plots (lower panels), showing consecutive injections of (A) apo mH0ACPa and (B) holo mH0ACPa, both against mKRb in the presence of NADPH. Thermogram traces for dilution control experiments are displayed in the upper panels in red.

Similar articles

Cited by

References

    1. Weissman KJ, Leadlay PF. Combinatorial bioisynthesis of reduced polyketides. Nature Rev. Microbiol. 2009;3:925–936. doi: 10.1038/nrmicro1287. - DOI - PubMed
    1. Keatinge-Clay AT. The structures of type I polyketide synthases. Nat. Prod. Rep. 2012;29:1050–1073. doi: 10.1039/c2np20019h. - DOI - PubMed
    1. Mercer AC, Burkhart MD. The ubiquitous carrier protein – a window to metabolite biosynthesis. Nat. Prod. Rep. 2006;24:750–773. doi: 10.1039/b603921a. - DOI - PubMed
    1. Crosby J, Crump MP. The structural role of the carrier protein - active controller or passive carrier. Nat. Prod. Rep. 2012;29:1111–1137. doi: 10.1039/c2np20062g. - DOI - PubMed
    1. Weissman KJ. Uncovering the structures of modular polyketide synthases. Nat. Prod. Rev. 2016;32:436–453. doi: 10.1039/C4NP00098F. - DOI - PubMed

Publication types

MeSH terms

Substances