Mutational analysis of peptidyl carrier protein and acyl carrier protein synthase unveils residues involved in protein-protein recognition.
4'-Phosphopantetheinyl transferases (PPTases) are essential for the production of fatty acids by fatty acid synthases (primary metabolism) and natural products by nonribosomal peptide synthetases and polyketide synthases (secondary metabolism). These systems contain carrier proteins (CPs) for the covalent binding of reaction intermediates during synthesis. PPTases transfer the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto conserved serine residues of the apo-CPs to convert them to their functionally active holo form. In bacteria, two types of PPTases exist that are evolutionary related but differ in their substrate spectrum. Acyl carrier protein synthases (AcpSs) recognize CPs from primary metabolism, whereas Sfp- (surfactin production-) type PPTases have a preference for CPs of secondary metabolism. Previous investigations showed that a peptidyl carrier protein (PCP) of secondary metabolism can be altered to serve as substrate for AcpS. We demonstrate here that a single mutation in PCP suffices for the modification of this CP by AcpS, and we have identified by mutational analysis several other PCP residues and two AcpS residues involved in substrate discrimination by this PPTase. These altered PCPs were still capable of serving their designated function in NRPS modules, and selective use of AcpS or Sfp leads to production of two different products by a trimodular NRPS.
Finking
Robert
R
Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
Mofid
Mohammad Reza
MR
Marahiel
Mohamed A
MA
D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases; D011485:Protein Binding; D013379:Substrate Specificity; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C082662:phosphopantetheinyl transferase; C073125:acpS protein, E coli; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase