title: Depsipeptide Intermediates Interrogate Proposed Biosynthesis of Cereulide, the Emetic Toxin of Bacillus cereus.
abstract: Cereulide and isocereulides A-G are biosynthesized as emetic toxins by Bacillus cereus via a non-ribosomal peptide synthetase (NRPS) called Ces. Although a thiotemplate mechanisms involving cyclo-trimerization of ready-made D-O-Leu-D-Ala-L-O-Val-L-Val via a thioesterase (TE) domain is proposed for cereulide biosynthesis, the exact mechanism is far from being understood. UPLC-TOF MS analysis of B. cereus strains in combination with (13)C-labeling experiments now revealed tetra-, octa-, and dodecapeptides of a different sequence, namely (L-O-Val-L-Val-D-O-Leu-D-Ala)1-3, as intermediates of cereulide biosynthesis. Surprisingly, also di-, hexa-, and decadepsipeptides were identified which, together with the structures of the previously reported isocereulides E, F, and G, do not correlate to the currently proposed mechanism for cereulide biosynthesis and violate the canonical NRPS biosynthetic logic. UPLC-TOF MS metabolite analysis and bioinformatic gene cluster analysis highlighted dipeptides rather than single amino or hydroxy acids as the basic modules in tetradepsipeptide assembly and proposed the CesA C-terminal C* domain and the CesB C-terminal TE domain to function as a cooperative esterification and depsipeptide elongation center repeatedly recruiting the action of the C* domain to oligomerize tetradepsipeptides prior to the release of cereulide from the TE domain by macrocyclization.
authors.affiliation: Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Straße 34, Freising, 85354, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Marxen
authors.affiliation: Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Straße 34, Freising, 85354, Germany.
authors.forename: Timo D
authors.initials: TD
authors.lastname: Stark
authors.affiliation: Department of Microbiology, Central Institute for Food and Nutrition Research, Technische Universität München, Freising, 85350.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rütschle
authors.affiliation: Department of Microbiology, Central Institute for Food and Nutrition Research, Technische Universität München, Freising, 85350.
authors.forename: Genia
authors.initials: G
authors.lastname: Lücking
authors.affiliation: Institute of Microbiology Department of Pathobiology, Functional Microbiology, University of Veterinary Medicine Vienna, Vienna, 1210, Austria.
authors.forename: Elrike
authors.initials: E
authors.lastname: Frenzel
authors.affiliation: Department of Microbiology, Central Institute for Food and Nutrition Research, Technische Universität München, Freising, 85350.|Department of Biosciences, Chair of Microbial Ecology, WZW, Technische Universität München, Freising, 85350, Germany.
authors.forename: Siegfried
authors.initials: S
authors.lastname: Scherer
authors.affiliation: Institute of Microbiology Department of Pathobiology, Functional Microbiology, University of Veterinary Medicine Vienna, Vienna, 1210, Austria.
authors.forename: Monika
authors.initials: M
authors.lastname: Ehling-Schulz
authors.affiliation: Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Straße 34, Freising, 85354, Germany.|Bavarian Center for Biomolecular Mass Spectrometry, Technische Universität München, Gregor-Mendel Strasse 4, 85354, Freising, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hofmann
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D047630:Depsipeptides / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D047630:Depsipeptides; C089294:cereulide; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase