Abstract
The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Bacterial Proteins
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Base Sequence
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Chymotrypsin
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Cloning, Molecular
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Cytochrome P-450 Enzyme System / genetics*
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Cytochrome P-450 Enzyme System / isolation & purification
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Escherichia coli / enzymology
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Escherichia coli / genetics*
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Mixed Function Oxygenases / genetics*
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Mixed Function Oxygenases / isolation & purification
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Nocardiaceae / enzymology*
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Nocardiaceae / genetics
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Oligonucleotide Probes
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Open Reading Frames
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Peptide Fragments / isolation & purification
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Polymerase Chain Reaction
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Promoter Regions, Genetic
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Restriction Mapping
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Thiolester Hydrolases / biosynthesis
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Thiolester Hydrolases / genetics*
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Thiolester Hydrolases / isolation & purification
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Trypsin
Substances
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Bacterial Proteins
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Oligonucleotide Probes
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Peptide Fragments
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Recombinant Proteins
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Cytochrome P-450 Enzyme System
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Mixed Function Oxygenases
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eryF protein, Saccharopolyspora erythraea
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Thiolester Hydrolases
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oleoyl-(acyl-carrier-protein) hydrolase
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Chymotrypsin
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Trypsin