Abstract
Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.
Highlights
Cyclic depsipeptides are natural products made of diverse acyl moieties linked by amide and ester bonds (Fig 1C and 1D) which have a broad range of biological and medical activities
The cereulide toxin (Fig 1D) is the causative agent in severe food poisoning associated with emetic strains of Bacillus cereus, which can lead to acute liver failure and death [9,10,11]
The cereulide synthetase subunits can be expressed in Escherichia coli and purified to homogeneity
Summary
Cyclic depsipeptides are natural products made of diverse acyl moieties linked by amide and ester bonds (Fig 1C and 1D) which have a broad range of biological and medical activities. Quorum sensing modulators, toxins and antibiotics [1,2,3]. The cereulide toxin (Fig 1D) is the causative agent in severe food poisoning associated with emetic strains of Bacillus cereus, which can lead to acute liver failure and death [9,10,11]. The main mechanism of cereulide poisoning is an emetic syndrome mediated by the serotonin 5-HT3 receptor and by stimulation of the vagus afferent nerve [12]. This toxin exerts cytotoxicity by disrupting the mitochondrial membrane potential through its K+ ionophoric activity [13]. Cereulide is highly resistant to heat, extreme pH, and proteases and remains a health
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