title: Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism.
abstract: The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases). They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs. PPTases, based on sequence and substrate specifity, have been classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism. The recently solved crystal structures of AcpS and Sfp-type PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) motif and similarities in CoA binding and polymerization mode. However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal. In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP. That, in turn, acts as a nucleophile to attack the beta-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (residues T111-S124) in Sfp is the PCP binding region. Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.
authors.affiliation: Fachbereich Chemie/Biochemie, Philipps-Universität Marburg, 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: Lars Oliver
authors.initials: LO
authors.lastname: Essen
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D003065:Coenzyme A / Q000737:chemistry; D004252:DNA Mutational Analysis; D055666:Lipopeptides; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics; D019216:Metals, Heavy / Q000737:chemistry; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000235:genetics*
chemical_list: D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D002413:Cations, Divalent; D055666:Lipopeptides; D008074:Lipoproteins; D019216:Metals, Heavy; D010456:Peptides, Cyclic; C082662:phosphopantetheinyl transferase; C009365:surfactin peptide; D017855:Transferases (Other Substituted Phosphate Groups); D003065:Coenzyme A