Bacterial cytochrome P450 system catabolizing the Fusarium toxin deoxynivalenol

Abstract

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (k(cat)/K(m)) of 6.4 mM(-1) s(-1). The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

Fig 1

Characterization of strain KSM1. (A) Phylogenetic analysis of strain KSM1 based on the 16S rRNA gene sequence. The 1,235 bp of the 16S rRNA gene of strain KSM1 was 96.4% identical to that of Sphingomonas wittichii strain RW1. The tree was constructed by the maximum likelihood method. Branches corresponding to partitions reproduced in less than 70% bootstrap replicates are collapsed. The GenBank accession numbers of the sequences are shown in parentheses. Sinorhizobium meliloti 1021 was used as an outgroup. Bootstrap values shown at the branch points are based on 1,000 resamplings (only values of >50% are shown). The bar indicates 0.02 substitutions per nucleotide position. (B) Assimilation of DON and NIV by strain KSM1. KSM1 cells were inoculated by adding 5 μl of stationary-phase seed cultures (OD₆₀₀ ≈ 0.08 in 1/10R2A liquid medium) to 5 ml of LSM. The values given are the means for three biological replicates. The error bars represent the standard deviations. The cell densities with different letters were significantly different (P < 0.05 by the Tukey honestly significant difference test). (C) Assimilation of 16-hydroxy-DON (16-HDON) by strain KSM1 in LSM containing 1% DMSO and 338 μM 16-HDON. The values given are the means for three biological replicates. The error bars represent the standard deviations. The cells were inoculated by adding 5 μl of stationary-phase seed cultures (OD₆₀₀ ≈ 0.08 in 1/10R2A liquid medium) to 5 ml of LSM.

Fig 2

UV-visible absorbance spectra of DdnA. Red, oxidized DdnA; blue, sodium dithionite-reduced DdnA; yellow, reduced DdnA saturated with carbon monoxide.

Fig 3

Mitochondrial-type [2Fe-2S] Fdxs and FAD-dependent FdRs among KSM1 and its relative bacteria. The enzymes of strain KSM1 and four KSM1 relatives (Sphingobium japonicum UT26, Sphingopyxis alaskensis RB2256, Novosphingobium aromaticivorans DSM12444, and Sphingomonas wittichii RW1) are shown with enzymes with genetically or biochemically characterized functions. The specific names of Fdxs and FdRs are shown in parentheses, if available. The numbers of the enzymes in panels A and C correspond to the sequence numbers of panel B and D, respectively. (A and C) Phylogenetic relationship between mitochondrial-type [2Fe-2S] Fdxs (A) and FAD-dependent FdRs (C). The trees were constructed by the maximum likelihood method. P450-Fdx, Fdx shown to act with P450 enzymes; ISC-Fdx, Fdx shown to be involved in iron-sulfur cluster biogenesis; P450-FdR, FdR shown to act with cytochrome P450 enzymes; RO-FdR, FdR shown to act with class IIB Rieske dioxygenases. The bars indicate 0.2 substitutions per amino acid position. (B and D) Conserved motifs among P450-Fdxs (B) and P450-FdRs (D). The amino acid positions conserved among >70% of the sequences were shaded with the Boxshade program. The arrows indicate the sites and directions of the degenerate primers used for amplification of the Kdx gene or KdR gene. Red triangle, alanine residue conserved among cluster I Fdxs and P450-Fdxs; open triangles, conserved Fe-S cluster-binding cysteine residues.

Fig 4

Mass spectra of compound A (16-HDON) (A) and DON (B) analyzed by negative-ion-mode LC-ESI-MS. cps, counts per second.

Fig 5

Schematic of DON hydroxylation by the DdnA-Kdx-KdR catalytic system.

Fig 6

Hydroxylation of DON mediated by DdnA reduced the inhibition of wheat seedlings. The wheat cultivar used was Norin no. 61 (moderately resistant to FHB) (61). Each seedling (n = 15) was grown from germinated grains for 7 days on gellan gum-soft gel medium (GSGM) containing DON or 16-HDON and 1% DMSO at 28°C. After incubation, the seedlings were cut off the grains, and the dry weight and length of the seedlings were measured. (A) Wheat seedlings grown on GSGM containing DON or 16-HDON. The white letters show the tested substrates and their concentrations (micromolar). Control seedlings were grown on GSGM containing 1% DMSO. All of the 15 seedlings tested for each treatment are shown. Bar, 100 mm. (B and C) Box plot representation of the dry weights (B) and lengths (C) of the seedlings shown in panel A. Medians (solid lines) and means (dashed lines) are shown in each box. The bottom and top of the box indicate the 25th and 75th percentiles, respectively. The bottom and top of the error bars represent the 10th and 90th percentiles, respectively. The diamonds represent the minimum and maximum values outside the error bars. The data were evaluated by Kruskal-Wallis ANOVA, followed by pairwise comparisons using the Mann-Whitney test with Bonferroni's adjustment. The dry weight and length indicated with different letters are significantly different (α = 0.05/21 = 0.00238 after Bonferroni's adjustment).