An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli.
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.
Caffrey
P
P
Department of Biochemistry, University of Cambridge, England.
Green
B
B
Packman
L C
LC
Rawlings
B J
BJ
Staunton
J
J
Leadlay
P F
PF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002918:Chymotrypsin; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009618:Nocardiaceae / Q000201:enzymology* / Q000235:genetics; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013869:Thiolester Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014357:Trypsin
D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase; D002918:Chymotrypsin; D014357:Trypsin