An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli

Eur J Biochem. 1991 Feb 14;195(3):823-30. doi: 10.1111/j.1432-1033.1991.tb15771.x.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins
  • Base Sequence
  • Chymotrypsin
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / isolation & purification
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / isolation & purification
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nocardiaceae / enzymology*
  • Nocardiaceae / genetics
  • Oligonucleotide Probes
  • Open Reading Frames
  • Peptide Fragments / isolation & purification
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Restriction Mapping
  • Thiolester Hydrolases / biosynthesis
  • Thiolester Hydrolases / genetics*
  • Thiolester Hydrolases / isolation & purification
  • Trypsin

Substances

  • Bacterial Proteins
  • Oligonucleotide Probes
  • Peptide Fragments
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • eryF protein, Saccharopolyspora erythraea
  • Thiolester Hydrolases
  • oleoyl-(acyl-carrier-protein) hydrolase
  • Chymotrypsin
  • Trypsin