Characterization of Cereulide Synthetase, a Toxin-Producing Macromolecular Machine

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.

Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels

“…Hitherto, research on the cereulide biosynthetic ces operon focused on the molecular and biochemical characterization of the non-ribosomal cereulide peptide synthetase encoded by cesA and cesB ( Ehling-Schulz et al, 2005b ; Magarvey et al, 2006 ; Alonzo et al, 2015 ; Marxen et al, 2015b ). However, little is known about the adjacent genes cesH, cesP, cesC , and cesD , which code for a putative hydrolase, a PPtase and a putative transport system of the ABC-type, respectively ( Ehling-Schulz et al, 2006 ).…”

“…The cesPTABCD genes represent an operon being transcribed as a single 23-kb polycistronic mRNA, whereas the adjacently located cesH is transcribed from its own promoter ( Dommel et al, 2010 ; Figure 1 ). The structural NRPS genes cesA and cesB encode the modules that are responsible for the activation and incorporation of each two monomers: The CesA2 and CesB2 submodules install D-alanine and L-valine, respectively, whereas CesA1 and CesB1 bind α-keto acids of leucine and valine (α-ketoisocaproic acid and α-ketoisovaleric acid; Magarvey et al, 2006 ; Alonzo et al, 2015 ). A central condensation domain in the C-terminal part of CesA together with a type I thioesterase (TE) domain located in the C-terminal part of CesB is suggested to act as esterification and elongation center before the final dodecadepsipeptide cereulide is released from the TE domain by macrocyclization ( Marxen et al, 2015b ).…”

Manipulation of Transmembrane Transport by Synthetic K+ Ionophore Depsipeptides and Its Implications in Glucose‐Stimulated Insulin Secretion in β‐Cells

“…Because B. cereus is a ubiquitous bacterial agent in nature, its spores can be found as a natural contaminant in a number of food products, and so the presence of cereulide at low concentrations is an undetected risk to public health. It is currently assumed that cereulide disrupts the transmembrane potential in the mitochondria of eukaryotic cells, leading to their degenerescence . The higher toxicity (about 15 times more toxic) of cereulide [ d ‐ O ‐Leu‐D‐Ala‐ l ‐ O ‐Val‐ l ‐Val] 3 relative to valinomycin [ d ‐ O ‐Hyi‐ d ‐Val‐ l ‐ O ‐Lac‐ l ‐Val] 3 is claimed to be due to a higher affinity for K + at physiological plasma concentration.…”

“…[1] Whereas valinomycin is ab iosynthetic product currently used as an antibiotic, [2] cereulide is an emetic toxin produced by specific strainso fBacillusc ereus, [3] which is ac ommon cause of several diseases. [4] Because B. cereus is au biquitous bacterial agent in nature, [5] its spores can be found as an aturalc ontaminant in an umber of food products, [6] and so the presence of cereulidea tl ow concentrations is an undetected risk to public health. It is currently assumed that cereulide disruptst he transmembrane potential in the mitochondria of eukaryotic cells, leadingt ot heir degenerescence.…”

“…It is currently assumed that cereulide disruptst he transmembrane potential in the mitochondria of eukaryotic cells, leadingt ot heir degenerescence. [4] The higher toxicity( about 15 times more toxic [7] )o f cereulide [d-O-Leu-D-Ala-l-O-Val-l-Val] 3 3 is claimed to be due to ah igher affinity [8] for K + at physiological plasma concentration. Althought he ingestion of high doses of cereulide causes acute emetic toxicity,v ery low doses of cereulide also exert toxicity, [9] with pancreatic beta cells (insulin-producing cells) being particularly sensitive.…”