Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin-producing polyketide synthase.
6-Deoxyerythronolide B synthase 3 (DEBS 3) is proposed to catalyse the fifth and sixth condensation cycles in the assembly of the polyketide 6-deoxyerythronolide B, the first isolatable intermediate in the biosynthesis of erythromycin A by Saccharopolyspora erythraea. The gene encoding DEBS 3 has previously been cloned and sequenced, and the deduced product is predicted to house nine fatty acid synthase-like activities on a 330-kDa polypeptide chain. The gene has been engineered into a pT-7-based expression system for over-expression in Escherichia coli. Recombinant DEBS 3 was found to constitute, after induction, 1-2% of soluble intracellular protein. DEBS 3 was purified from extracts of the recombinant E. coli to apparent homogeneity, and was found not to be modified by covalent attachment of the prosthetic group 4'-phosphopantetheine. Incubation with (R,S)-methylmalonyl-CoA, the presumed source of extension units for polyketide chain assembly, led to hydrolysis of the thioester, implying that the methylmalonyl-CoA:ACP acyltransferase domains in DEBS 3 are correctly folded and able to catalyse this side-reaction. During this reaction, DEBS 3 became transiently radiolabelled, consistent with the intermediacy of an acylenzyme. The native molecular mass of the protein by gel filtration chromatography was 668 kDa which corresponds either to a dimer or to a highly asymmetric monomer.
Cambridge Centre for Molecular Recognition, University of Cambridge, England.
G A
GA
Roberts
J
J
Staunton
P F
PF
Leadlay
D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004917:Erythromycin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics
D000214:Acyl Coenzyme A; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C015357:methylmalonyl-coenzyme A; D004917:Erythromycin