The enzymatic epimerization of deoxynivalenol by Devosia mutans proceeds through the formation of 3-keto-DON intermediate

Abstract

The enzymatic detoxification of deoxynivalenol (DON) is a promising mitigation strategy for addressing this mycotoxin contamination of cereal grains. A recently described bacterium, Devosia mutans 17-2-E-8, capable of transforming DON into its non-toxic stereoisomer 3-epi-DON, holds promise for the development of such applications. Earlier observations suggested that DON epimerization proceeds via a two-step catalysis with 3-keto-DON as an intermediate. The results of this study indicate that NADPH is required for DON epimerization by cell-free protein extracts of D. mutans, while high concentrations of glucose and sucrose have a suppressive effect. Chemically synthesized 3-keto-DON incubated with D. mutans protein fractions enriched by ammonium sulfate precipitation at 35-55% saturation selectively reduced 3-keto-DON to 3-epi-DON, but fell short of supporting the complete epimerization of DON. In addition, seven Devosia species investigated for DON epimerization were all able to reduce 3-keto-DON to 3-epi-DON, but only a few were capable of epimerizing DON. The above observations collectively confirm that the enzymes responsible for the oxidation of DON to 3-keto-DON are physically separate from those involved in 3-keto-DON reduction to 3-epi-DON. The enzymatic nature of DON epimerization suggests that the process could be used to develop genetically engineered crops or microorganisms, ultimately reducing foodborne exposure of consumers and farm animals to DON.

Figure 1

The two-step bacterial transformations of deoxynivalenol (DON) to 3-epi-DON proceeds through the formation of 3-keto-DON intermediate. DON biotransformation to 3-epi-DON in different Gram positive and Gram negative bacterial isolates (including Devosia mutans) is predicted to proceed through two separate enzymatic steps. The first oxidation step leads to the formation of 3-keto-DON intermediate while the second reduction step leads to 3-epi-DON accumulation.

Figure 2

The chemical synthesis of 3-keto-DON and the retrieved proton (¹H) NMR spectra of the synthesized compound. (Top): Chemical synthesis of 3-keto-DON (D) encompasses protecting the 7,15-dihydroxyl groups of DON (A) by using 2,2-dimethoxypropane (i) before oxidizing the secondary hydroxyl (ii) in a later phase. The protecting-groups were removed later through a hydrolysis initiated with TFA (iii) to finally obtain the desired 3-keto-DON intermediate (D). (Bottom): The obtained chemical shifts of ¹H-NMR signals of the chemically synthesized 3-keto-DON are identical with previously published data for the same compound^, . A detailed discussion of the spectra is found within the results section. An overall yield close to 36% was achieved combined with purity levels close to 95%.

Figure 3

The chromatographic separation of DON, 3-keto-DON and 3-epi-DON by LC-MS/MS and DON epimerization dynamics in Devosia mutans 17-2-E-8 cultures growing in Corn Meal broth (CMB). (Top): The chromatographic analysis of DON, 3-keto-DON and 3-epi-DON using LC-MS/MS platform. The extracted ion chromatograms for transitions m/z 296.5 > 249.1 for DON as well as 3-epi-DON and m/z 294.9 > 247.1 for 3-keto-DON are shown. (Bottom): Both the disappearance of DON and the appearance/accumulation of 3-keto-DON and 3-epi-DON were monitored in D. mutans cultures up to 42 h as reported within the Materials and Methods section.

Figure 4

The detection of 3-keto-DON in Devosia mutans 17-2-E-8 bacterial cultures and 3-keto-DON and 3-epi-DON concurrence during Devosia mutans 17-2-E-8 assisted-epimerisation of DON. (Top): The chromatographic separation of 3-keto-DON obtained from depleted cultures of Devosia mutans 17-2-E-8 at 0 h, 18 h, and 36 h (panel B, C, and D) in comparison to 3-keto-DON standard that was chemically synthesized (panel A). The analysis was conducted using LC-MS/MS by monitoring mass transition m/z 294.9 > 247.1 as described in the Materials and Methods section. (Bottom): Different media preparations containing: glucose, sucrose and corn starch (carbon sources); corn steep liquor, peptone, yeast extract, and urea (organic nitrogen sources); ammonium sulfate and ammonium nitrate (inorganic nitrogen sources) were tested for DON epimerization. Every broth that supported DON epimerization showed the concurrence of 3-keto-DON at the same time. While experiments were conducted in triplicates to confirm the observed tendencies, no quantitative statistical inferences between different broths are suggested.

Figure 5

DON epimerization in cell-free lysates of Devosia mutans and the enzymatic activity of selected ammonium sulfate protein fractions of Devosia mutans. (Top): Whole cell-free lysates of bacteria (n = 5) were incubated for two hours with DON (as substrate) without any added cofactors or with NADP(H) at 100 µM. Concentrations of DON and 3-epi-DON were determined by HPLC-MS/MS methods. (Bottom): The 35–55% ammonium sulfate protein fraction prepared as reported earlier in the Materials and Methods section was tested for the enzymatic activity by adding DON or 3-keto-DON (as substrates) separately with a source of NADP(H). After an overnight incubation, the concentrations of DON, 3-keto-DON and 3-epi-DON were determined by HPLC-MS/MS methodology. While experiments were conducted in triplicates to confirm the observed tendencies, no quantitative statistical inferences between reactions are suggested for the bottom part of the figure.

Figure 6

The ability of different Devosia type-strains to epimerize DON or to selectively reduce 3-keto-DON to 3-epi-DON. The simultaneous oxidation and reduction of the C3 carbon (epimerization) in DON (Top) or the selective reduction of 3-keto-DON to 3-epi-DON (Bottom) were tested after growing the pure isolates within the respective media preparations recommended by type-strains suppliers (DMSZ & ATCC culture collections). The concentrations of DON, 3-keto-DON, and 3-epi-DON were monitored as reported within the Materials and Methods section (after 24 h incubations). While experiments were conducted in triplicates to confirm the observed tendencies, no quantitative statistical inferences between strains are suggested. The incorporated arrows highlight the presence of traces of different DON metabolites.

Figure 7

Molecular modeling of DON and 3-epi-DON. The molecular modeling of DON (right panel) and 3-epi-DON (left panel) chemical structures was achieved by Hyperchem 8.0. The decreased distance between the hydrogen atom of the C3 hydroxyl group and the oxygen atom of the epoxide group in 3-epi-DON (2.5 Å) in comparison to DON (3.1 Å) suggests the possibility of an intramolecular hydrogen bond formation that contributes to 3-epi-DON stability and influences its ribosomal binding capacity.