title: A mechanism based protein crosslinker for acyl carrier protein dehydratases.
abstract: Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (approximately 9 kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4'-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP-DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, United States.
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Meier
authors.forename: Robert W
authors.initials: RW
authors.lastname: Haushalter
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D064429:Fatty Acid Synthases / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D048630:Polyketide Synthases / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D003432:Cross-Linking Reagents; D048630:Polyketide Synthases; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases