Abstract
The emetic Bacillus cereus toxin cereulide presents an enormous safety hazard in the food industry, inducing emesis and nausea after the consumption of contaminated foods. Additional to cereulide itself, seven structurally related isoforms, namely the isocereulides A–G, have already been elucidated in their chemical structure and could further be identified in B. cereus contaminated food samples. The newly performed isolation of isocereulide A allowed, for the first time, 1D- and 2D-NMR spectroscopy of a biosynthetically produced isocereulide, revealing results that contradict previous assumptions of an l-O-Leu moiety within its chemical structure. By furthermore applying posthydrolytical dipeptide analysis, amino acid and α-hydroxy acid analysis by means of UPLC-ESI-TOF-MS, as well as MSn sequencing, the structure of previously reported isocereulide A could be corrected. Instead of the l-O-Leu as assumed to date, one l-O-Ile unit could be verified in the cyclic dodecadepsipeptide, revising the structure of isocereulide A to [(d-O-Leu-d-Ala-l-O-Val-l-Val)2(d-O-Leu-d-Ala-l-O-Ile-l-Val)].
Highlights
The ubiquitous, endospore-forming, facultative anaerobe bacterium Bacillus cereus [1] is commonly known as a food-borne pathogen, causing, among others, emesis in consequence of the production of its emetic toxin cereulide (1), which is inert to a wide range of environmental parameters such as temperature, pH values, and enzymes [2,3,4]
The characteristic dodecadepsipeptide structure of cereulide (1) is composed of the three times circularly repeating tetradepsipeptide unit L-O-Val-L-Val-D-O-Leu-D-Ala, leading to a rectangular cylindrical shape [5,6,7,8]. This complex three-dimensional dodecadepsipeptide structure is biosynthetically assembled by nonribosomal peptide synthetases (NRPSs), designated CesNRPS [9]
Apart from the structural cereulide synthetase genes cesA and cesB, the ces gene locus comprises a phosphopantheteintransferase involved in the activation of the NRPS machinery, a type II thioesterase with a proofreading function and an ABC transporter, recently shown to be involved in cereulide export and directly in cereulide biosynthesis [12,13,14]
Summary
The ubiquitous, endospore-forming, facultative anaerobe bacterium Bacillus cereus [1] is commonly known as a food-borne pathogen, causing, among others, emesis in consequence of the production of its emetic toxin cereulide (1), which is inert to a wide range of environmental parameters such as temperature, pH values, and enzymes [2,3,4]. The characteristic dodecadepsipeptide structure of cereulide (1) is composed of the three times circularly repeating tetradepsipeptide unit L-O-Val-L-Val-D-O-Leu-D-Ala, leading to a rectangular cylindrical shape [5,6,7,8] This complex three-dimensional dodecadepsipeptide structure is biosynthetically assembled by nonribosomal peptide synthetases (NRPSs), designated CesNRPS [9]. Cereulide (1) biosynthesis takes place over a generated L-O-Val-L-Val-D-O-Leu-D-Ala-PCP-coupled intermediate [8] or via the trimerization and macrocyclization of an L-O-Val-L-Val-D-O-Leu-D-Ala intermediate by the thioesterase (TE)-dependent CesTE. The latter hypothesis aligns the biosynthesis pathway with the structurally similar toxin valinomycin [17,18]. After full structure characterization of the aforementioned isocereulide by means of UPLC-TOF-MS experiments, ion-trap MSn sequencing, posthydrolytic dipeptide, and enantioselective amino acid analysis, as well as further structure investigation regarding the α-hydroxy acid composition, a corrected chemical structure could be elucidated, and the chemical structure of isocereulide A (2) was revised
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