Abstract
Acyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis. The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP. Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine. The presence of this intramolecular disulphide was confirmed by tryptic digestion of the ACP followed by ESMS analysis of the fragments. An act Cys17Ser ACP was engineered by site-directed mutagenesis. S-Acyl adducts of act C17S, oxytetracycline and griseusin holo ACPs were rapidly formed by reaction with hexanoyl, 5-ketohexanoyl and protected acetoacetyl imidazolides. Comparisons with type 11 FAS ACPs were made.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acyl Carrier Protein / chemistry
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Acyl Carrier Protein / genetics
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Acyl Carrier Protein / metabolism*
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Acylation
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Chromatography, High Pressure Liquid
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Cysteine / metabolism
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Disulfides / metabolism
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Escherichia coli / genetics
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Mass Spectrometry
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Multienzyme Complexes / chemistry
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Multienzyme Complexes / genetics
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Multienzyme Complexes / metabolism*
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Mutagenesis, Site-Directed
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Peptide Fragments / analysis
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Recombinant Proteins
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Streptomyces / enzymology*
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Transferases (Other Substituted Phosphate Groups) / metabolism
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Trypsin / metabolism
Substances
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Acyl Carrier Protein
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Disulfides
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Multienzyme Complexes
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Peptide Fragments
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Recombinant Proteins
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Transferases (Other Substituted Phosphate Groups)
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holo-(acyl-carrier-protein) synthase
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Trypsin
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Cysteine