Identification of a phosphopantetheinyl transferase for erythromycin biosynthesis in Saccharopolyspora erythraea.
Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins (CPs) from fatty acid synthases (FASs) (primary metabolism), polyketide synthases (PKSs), and non-ribosomal polypeptide synthetases (NRPSs) (secondary metabolism). Bacteria typically harbor one PPTase specific for CPs of primary metabolism ("ACPS-type" PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism ("Sfp-type" PPTases). In order to identify the PPTase(s) associated with erythromycin biosynthesis in Saccharopolyspora erythraea, we have used the genome sequence of this organism to identify, clone, and express (in Escherichia coli) three candidate PPTases: an ACPS-type PPTase (S. erythraea ACPS) and two Sfp-type PPTases (a discrete enzyme (SePptII) and another that is integrated into a modular PKS subunit (SePptI)). In vitro analysis of these recombinant PPTases, with an acyl carrier protein-thioesterase (ACP-TE) didomain from the erythromycin PKS as substrate, revealed that only SePptII is active in phosphopantetheinyl transfer with this substrate. SePptII was also shown to provide complete modification of ACP-TE and of an entire multienzyme subunit from the erythromycin PKS in E. coli. The efficiency of the SePptII in phosphopantetheinyl transfer in E. coli makes it an attractive alternative to other Sfp-type PPTases for co-expression experiments with PKS proteins.
Weissman
Kira J
KJ
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK. kjw21@cus.cam.ac.uk
Hong
Hui
H
Oliynyk
Markiyan
M
Siskos
Alexis P
AP
Leadlay
Peter F
PF
D000213:Acyl Carrier Protein / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002853:Chromatography, Liquid; D003062:Codon / Q000235:genetics; D004917:Erythromycin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D016681:Genome, Fungal; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011492:Protein Hydrolysates / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
D000213:Acyl Carrier Protein; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003062:Codon; D009097:Multienzyme Complexes; D011492:Protein Hydrolysates; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D004917:Erythromycin; D017855:Transferases (Other Substituted Phosphate Groups); D014357:Trypsin