title: Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains.
abstract: Here we report a new experimental approach to characterize recombinant nonribosomal peptide cyclases that do not show activity with conventional N-acetylcysteamine (SNAC) substrates. To explore the great potential of these domains for the catalysis of cell-free cyclization reactions, the new strategy takes advantage of the direct interaction between the natural substrate where the peptide chain is attached to the phosphopantetheine arm of the peptidyl carrier protein (PCP) and the peptide cyclase. A prerequisite for this reaction is the promiscuity of the Bacillus subtilis phosphopantetheinyl transferase Sfp for loading chemically synthesized peptidyl-coenzyme A substrates instead of the smaller natural substrate coenzyme A (CoASH) onto apoPCP. With this novel method we were able to characterize the regioselectivity of branched-chain cyclization catalyzed by the fengycin cyclase, which displays no activity with peptidyl-SNAC substrates.
authors.affiliation: Fachbereich Chemie/Biochemie, Philipps-Univerität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Sieber
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D003065:Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D003500:Cyclization; D055666:Lipopeptides; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D055666:Lipopeptides; D008074:Lipoproteins; C082662:phosphopantetheinyl transferase; C049972:fengycin; D017855:Transferases (Other Substituted Phosphate Groups); D013869:Thiolester Hydrolases; D003065:Coenzyme A; D000111:Acetylcysteine