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J Am Chem Soc. 2012 Jan 18;134(2):769-72. doi: 10.1021/ja2082334. Epub 2011 Dec 29.

Dehydratase-specific probes for fatty acid and polyketide synthases.

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Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.


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.

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