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 13C-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.
Highlights
The endospore-forming bacterium Bacillus cereus is a food-borne human pathogen causing diarrhea due to the activity of haemolytic and nonhaemolytic enterotoxins as well as food intoxications induced by the heat-stable, emesis provoking cereulide[1,2]
Biosynthesis of fungal or bacterial cyclooligomer depsipeptides, such as the cyclohexadepsipeptide enniatin from Fusarium species or the cyclooctadepsipeptide bassianolide from Beauveria bassiana, as well as the cyclodecadepsipeptide valinomycin from Streptomyces species or the cyclododecadepsipeptide cereulide from B. cereus is performed by large multi-domain non-ribosomal peptide synthetases (NRPS)[5,6,7,8]
Chain elongation of the intermediate is mediated by a condensation (C) domain catalyzing the nucleophilic attack of the downstream peptidyl carrier protein (PCP)-bound acceptor monomer on the activated thioester of the upstream PCP-bound donor monomer[13], giving rise to the dipeptide intermediate D-O-Leu-D-Ala-S-PCP
Summary
Interrogate Proposed Biosynthesis received: 10 March 2015 accepted: 22 April 2015 Published: 27 May 2015 of Cereulide, the Emetic Toxin of Bacillus cereus. Cereulide and isocereulides A-G are biosynthesized as emetic toxins by Bacillus cereus via a nonribosomal peptide synthetase (NRPS) called Ces. 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. Chain elongation of the intermediate is mediated by a condensation (C) domain catalyzing the nucleophilic attack of the downstream PCP-bound acceptor monomer on the activated thioester of the upstream PCP-bound donor monomer[13], giving rise to the dipeptide intermediate D-O-Leu-D-Ala-S-PCP Due to their enantioselectivity, they are thought to act as selective filters, facilitating the production of NRPS products in a directed manner[14]. The ces gene locus was reinvestigated using bioinformatic approaches
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