title: Sulfonyl 3-alkynyl pantetheinamides as mechanism-based cross-linkers of acyl carrier protein dehydratase.
abstract: Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein-protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic β-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ishikawa
authors.forename: Robert W
authors.initials: RW
authors.lastname: Haushalter
authors.forename: D John
authors.initials: DJ
authors.lastname: Lee
authors.forename: Kara
authors.initials: K
authors.lastname: Finzel
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000378:metabolism; D000480:Alkynes / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D025941:Protein Interaction Mapping; D013450:Sulfones / Q000737:chemistry*
chemical_list: D000213:Acyl Carrier Protein; D000480:Alkynes; D003432:Cross-Linking Reagents; D013450:Sulfones; D064429:Fatty Acid Synthases