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Harnessing the Chemical Activation Inherent to Carrier Protein-Bound Thioesters for the Characterization of Lipopeptide Fatty Acid Tailoring Enzymes
journal contribution
posted on 2008-02-27, 00:00 authored by Florian Kopp, Uwe Linne, Markus Oberthür, Mohamed A. MarahielHere, we report a new experimental approach utilizing an amide ligation reaction for the
characterization of acyl carrier protein (ACP)-bound reaction intermediates, which are otherwise difficult to
analyze by traditional biochemical methods. To explore fatty acid tailoring enzymes of the calcium-dependent
antibiotic (CDA) biosynthetic pathway, this strategy enabled the transformation of modified fatty acids,
covalently bound as thioesters to an ACP, into amide ligation products that can be directly analyzed and
compared to synthetic standards by HPLC-MS. The driving force of the amide formation is the
thermodynamic activation inherent to thioester-bound compounds. Using this novel method, we were able
to characterize the ACP-mediated biosynthesis of the unique 2,3-epoxyhexanoyl moiety of CDA, revealing
a new type of FAD-dependent oxidase HxcO with intrinsic enoyl-ACP epoxidase activity, as well as a second
enoyl-ACP epoxidase, HcmO. In general, our approach should be widely applicable for the in vitro
characterization of other biosynthetic systems acting on carrier proteins, such as integrated enzymes from
NRPS and PKS assembly lines or tailoring enzymes of fatty and amino acid precursor synthesis.
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PKS assembly linesacid precursor synthesisbiosynthetic systemsenzymeamide formationChemical Activation Inherentamide ligation productsapproachcharacterizationepoxidaseACPCDAamide ligation reactionbiosynthetic pathwayLipopeptide FattyNRPScarrier proteinsTailoring EnzymesHerenovel methodacyl carrier protein
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