title: 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.
authors.affiliation: Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Shakya
authors.forename: Heriberto
authors.initials: H
authors.lastname: Rivera
authors.forename: D John
authors.initials: DJ
authors.lastname: Lee
authors.forename: Matt J
authors.initials: MJ
authors.lastname: Jaremko
authors.forename: James J
authors.initials: JJ
authors.lastname: La Clair
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Fox
authors.forename: Robert W
authors.initials: RW
authors.lastname: Haushalter
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Schaub
authors.forename: Joel
authors.initials: J
authors.lastname: Bruegger
authors.forename: Jesus F
authors.initials: JF
authors.lastname: Barajas
authors.forename: Alexander R
authors.initials: AR
authors.lastname: White
authors.forename: Parminder
authors.initials: P
authors.lastname: Kaur
authors.forename: Emily R
authors.initials: ER
authors.lastname: Gwozdziowski
authors.forename: Fiona
authors.initials: F
authors.lastname: Wong
authors.forename: Shiou-Chuan
authors.initials: SC
authors.lastname: Tsai
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007659:Ketones; D048630:Polyketide Synthases