title: Dehydratase-specific probes for fatty acid and polyketide synthases.
abstract: We targeted the development of a dehydratase (DH)-specific reactive probe that can facilitate detection, enrichment, and identification of DH enzymes in fatty acid synthases (FASs) and polyketide synthases (PKSs). The first reported mechanism-based inactivator, 3-decynoyl-N-acetylcysteamine (3-decynoyl-NAC), while active against the Escherichia coli β-hydroxydecanoyl thiol ester DH FabA, translates poorly to an activity-based probe because of nonspecific reactivity of the thioester moiety. Here we describe the design, synthesis, and utility of a DH-specific probe that contains a sulfonyl 3-alkyne reactive warhead engineered to avoid hydrolysis or nonenzymatic inactivation. When coupled with a fluorescent tag, this probe targets DH enzymes from recombinant type I and type II FAS and PKS enzyme systems and in whole proteomes. Activity studies, including FabA inactivation and antibiotic susceptibility, suggest that this sulfonyl 3-alkyne scaffold selectively targets a common DH mechanism. These studies indicate that the DH-specific mechanism-based probe can greatly accelerate both the functional characterization and molecular identification of virtually any type of FAS and PKS in complex proteomes.
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: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D054889:Fatty Acid Synthase, Type II / Q000235:genetics / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000378:metabolism*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D048630:Polyketide Synthases; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases; C047118:3-hydroxyacyl-(acyl-carrier-protein) dehydratase; D054889:Fatty Acid Synthase, Type II