The Identification of DepB: An Enzyme Responsible for the Final Detoxification Step in the Deoxynivalenol Epimerization Pathway in Devosia mutans 17-2-E-8

Abstract

Deoxynivalenol (DON) is one of the most common mycotoxins found in cereal grains and grains contaminated with DON can cause health issues for both humans and animals and result in severe economic losses. Currently there is no feasible method to remediate affected grains. The development of a biological method for detoxification is becoming increasingly more plausible with the discovery of microbes which can transform DON to a relatively non-toxic stereoisomer, 3-epi-DON. Although bacteria capable of detoxifying DON have been known for some time, it is only recently an enzyme responsible was identified. In Devosia mutans 17-2-E-8 (Devosia sp. 17-2-E-8) a two-step DON epimerization (Dep) pathway, designated as the Dep system, completes this reaction. DepA was recently identified as the enzyme responsible for the conversion of DON to 3-keto-DON, and in this report, DepB, a NADPH dependent dehydrogenase, is identified as the second and final step in the pathway. DepB readily catalyzes the reduction of 3-keto-DON to 3-epi-DON. DepB is shown to be moderately thermostable as it did not lose significant activity after a heat treatment at 55°C and it is amenable to lyophilization. DepB functions at a range of pH-values (5-9) and functions equally well in multiple common buffers. DepB is clearly a NADPH dependent enzyme as it utilizes it much more efficiently than NADH. The discovery of the final step in the Dep pathway may provide a means to finally mitigate the losses from DON contamination in cereal grains through an enzymatic detoxification system. The further development of this system will need to focus on the activity of the Dep enzymes under conditions mimicking industrially relevant conditions to test their functionality for use in areas such as corn milling, fuel ethanol fermentation or directly in animal feed.

Keywords: 3-epi-DON; 3-keto-DON; deoxynivalenol (DON); detoxification; mycotoxin; reduction.

FIGURE 1

The DON epimerization pathway. DON is oxidized to 3-keto-DON by DepA followed by a reduction to 3-epi-DON by DepB.

FIGURE 2

Genomic location of D. mutans depB region. (A) D. mutans genome atlas showing protein sequence similarities with 19 other Devosia strains. The inner three rings represent the forward CDS (green), reverse CDS (red) and contigs (gray). The remaining rings (red) represent the tblastn hits of the predicted proteins from 19 Devosia strains against D. mutans (inner to outer): D. mutans, Devosia sp. DBB001, Devosia sp. A16, D. chinhatensis, D. epidermidihirudinis, D. geojensis, Devosia sp. H5989, D. insulae, Devosia sp. LC5, Devosia sp. Leaf420, Devosia sp. Leaf64, D. limi, D. psychrophila, D. riboflavina, Devosia sp. Root105, Devosia sp. Root413D1, Devosia sp. Root436, Devosia sp. Root635, Devosia sp. Root685, D. soli. The region surrounding depB is magnified (outlined in black) and depB highlighted in blue. (B) Gene diagram of depB (red) and surrounding genes (blue, predicted function; black, hypothetical protein).

FIGURE 3

The pH dependency of DepB. Reactions contained 50 mM of three component buffer at various pH-values, 100μg ml^-1 3-keto-DON, 400μM NADPH, 4.7μg DepB, the reaction was stopped after 15 min.

FIGURE 4

DepB activity using different nicotinamide cofactors. Reactions contain 50 mM of Tris pH 7.5, 100μg ml^-1 3-keto-DON, 400μM NADPH or NADH, 4.7μg DepB and the reaction was allowed to proceed for 15 min or overnight before it was stopped with acidified methanol.

FIGURE 5

The effect of heat treatment on the activity of DepB. Each reaction contained 100μg ml^-1 3-keto-DON, 400μM NADPH, 5μg DepB in 50 mM of Tris pH 7.5. Treated samples were subjected to a 1 h incubation at temperature then cooled on ice. The activity of the enzyme after treatment was plotted relative to the activity to the treatment at 35°C. a, not significantly different from 35°C. b, not significantly different from 60°C. c, not significantly different from 65°C.

FIGURE 6

The effect of temperature on the activity of DepB. Each reaction contained 100μg ml^-1 3-keto-DON, 400μM NADPH, 5μg DepB in 50 mM of Tris pH 7.5. The activity of the enzyme at each temperature was plotted relative to the activity at 30°C.

FIGURE 7

The effect of lyophilization on the activity of DepB. Each reaction contained 100μg ml^-1 3-keto-DON, 400μM NADPH, 4.7μg DepB in 50 mM of Tris pH 7.5 and was stopped after 12 min. Activity is relative to DepB which was not lyophilized. Neg Ctl had no enzyme added to the solution.