title: Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation.
abstract: Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143.
authors: Kathryn D
authors: KD
authors: Bewley
authors: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602.
authors: Philip R
authors: PR
authors: Bennallack
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143.
authors: Mark A
authors: MA
authors: Burlingame
authors: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602.
authors: Richard A
authors: RA
authors: Robison
authors: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602.
authors: Joel S
authors: JS
authors: Griffitts
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143; susan.millerphd@ucsf.edu.
authors: Susan M
authors: 0000-0002-8221-930X
authors: SM
authors: Miller
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001430:Bacteriocins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D040141:Staphylococcaceae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001430:Bacteriocins; D010455:Peptides; C009779:micrococcin
keywords: RiPP; affinity-tagged intermediates; antibiotic; biosynthesis; thiopeptide