Fig 1.
Cereulide synthetase produces the emetic toxin cereulide.
(A and B) Modules CesA1 and CesB1 contain KR domains which catalyze the reduction of bound keto acyl groups. (C) Schematic diagram of cereulide synthetase and the synthesis of D-HIC—D-Ala—L-HIV—L-Val, which is trimerized to produce mature cereulide (D). (E) Denaturing and (F) native PAGE of NRPS proteins CesA and CesB. CesA and CesB migrate slightly faster than expected from their molecular masses. Domain abbreviations: A, adenylation; KR, ketoreductase; T, thiolation; C, condensation; E, epimerization; TE, thioesterase.
Fig 2.
Kinetic characterization of adenylation by A domains using the ATP-PPi exchange assay.
Initial velocity versus substrate concentration plots for adenylation of cognate substrates for CesA1 (A), CesA2 (B), CesB1 (C) and CesB2 (D). Curves were fit to the Michaelis-Menten equation. The kinetic parameters obtained are listed in Table 1.
Fig 3.
Kinetic characterization of adenylation by A domains using the pyrophosphate production assay.
Initial velocity versus substrate concentration plots for adenylation of cognate substrates for CesA1 (A), CesA2 (B), CesB1 (C) and CesB2 (D). Curves were fit to the Michaelis-Menten equation. The kinetic parameters obtained are listed in Table 1.
Table 1.
Apparent catalytic constants of adenylation by cereulide synthetase A domains using the ATP-PPi exchange and pyrophosphate production assays.
Fig 4.
The MbtH-like protein of B. cereus does not affect the adenylation reactions of cereulide synthetase.
The adenylation reaction of each A domain in CesA (A) and CesB (B) with the cognate substrate was evaluated by the ATP-PPi exchange assay at a single point of substrate, with or without MbtH in the reaction, in triplicate. Data is expressed in counts per minute (cpm).
Fig 5.
Evaluation of α-keto acid side chain selectivity of cereulide synthetase.
The ATP-PPi exchange assay was performed with CesA1 (A) and CesB1 (B) and various α-keto acids as substrates, in triplicate. See S1 Fig for a comprehensive list of the monomers, their abbreviations, and their structures. Data was normalized to the activity obtained with the cognate substrate.
Fig 6.
Vinylsulfonamide compounds inhibit α-keto acid adenylation.
(A) Structure of the vinylsulfonamide inhibitor for CesA1, α-hydroxyisocaproic acyl-vinylsulfonamide adenylate. The A domain should catalyze the nucleophilic attack of the pantetheine arm thiol on the vinylsulfonamide analogue, but the adenine analogue should not be released in the reaction, trapping the T domain with the A domain. (B) CesA1 is inhibited by the vinylsulfonamide inhibitor. CesA1 was incubated with DMSO or the inhibitor in DMSO. Excess inhibitor and/or DMSO was removed with a desalting column prior to ATP-PPi exchange assay using α-KIC.
Fig 7.
NADPH affinity and turnover in the KR domains in CesA1 and CesB1.
(A,B) Fluorescence intensity enhancement is plotted against protein concentration using a fixed concentration of NADPH (2.5 μM); λexc = 340nm and λemm = 445nm for CesA1 and CesB1. Curves were fitted to a Morrison equation that includes terms for enhancement of NADPH fluorescence upon binding. (C) Enzymatic NADPH consumption as reported by fluorescence intensity (λexc = 340nm and λemm = 445nm).
Fig 8.
LC-MS analysis of the peptide synthesis reaction of in vitro-reconstituted cereulide synthetase.
Extracted ion chromatograms (EIC), from (A) synthetic standards of dipeptides 1 and 2, and (B) from the products of a peptide synthesis reaction of CesA, CesB and substrates. EICs are extracted using exact m/z values (±0.05 mass units) calculated from the [M-H]- ions of the compounds shown in (C). Mass spectra corresponding to predominant peaks are shown as insets. (C) Putative chemical structures of the cereulide intermediates detected in the peptide synthesis reaction. The structures are consistent with exact masses and determined molecular formulas, as well as the elution profile of the authentic standards of dipeptides. The shown stereochemistry of the tetra- and octapeptides (3 and 4) are consistent with their precursor molecules (1 and 2) and their successor molecule (cereulide). However, these putative chemical structures are not absolutely proven here.
Table 2.
MS analysis of the peptide synthesis reaction of in vitro-reconstituted cereulide synthetase.