title: Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp.
abstract: The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.
authors.affiliation: Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Mofid
authors.forename: Robert
authors.initials: R
authors.lastname: Finking
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D012694:Serine; D010649:Phenylalanine; D011392:Proline; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D010453:Peptide Synthases; C035180:tyrocidine synthetase