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. 2018 Dec 12;9:1846.
doi: 10.3389/fpls.2018.01846. eCollection 2018.

Structure-Guided Mechanisms Behind the Metabolism of 2,4,6-Trinitrotoluene by Glutathione Transferases U25 and U24 That Lead to Alternate Product Distribution

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Structure-Guided Mechanisms Behind the Metabolism of 2,4,6-Trinitrotoluene by Glutathione Transferases U25 and U24 That Lead to Alternate Product Distribution

Kyriakos Tzafestas et al. Front Plant Sci. .
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Abstract

The explosive xenobiotic 2,4,6-trinitrotoluene (TNT) is a major worldwide environmental pollutant and its persistence in the environment presents health and environmental concerns. The chemical structure of TNT dictates that biological detoxification pathways follow predominantly reductive transformation of the nitro groups, and as a result, TNT is notoriously recalcitrant to mineralization in the environment. Plant-based technologies to remediate this toxic pollutant rely on a solid understanding of the biochemical detoxification pathways involved. Toward this, two Arabidopsis Tau class glutathione transferases, GSTU24 and GSTU25, have been identified that catalyze the formation of three TNT-glutathionylated conjugates. These two GSTs share 79% identity yet only GSTU25 catalyzes the substitution of a nitro group for sulfur to form 2-glutathionyl-4,6-dinitrotoluene. The production of this compound is of interest because substitution of a nitro group could lead to destabilization of the aromatic ring, enabling subsequent biodegradation. To identify target amino acids within GSTU25 that might be involved in the formation of 2-glutathionyl-4,6-dinitrotoluene, the structure for GSTU25 was determined, in complex with oxidized glutathione, and used to inform site-directed mutagenesis studies. Replacement of five amino acids in GSTU24 established a conjugate profile and activity similar to that found in GSTU25. These findings contribute to the development of plant-based remediation strategies for the detoxification of TNT in the environment.

Keywords: 2,4,6-trinitrotoluene; Arabidopsis; GST; TNT; detoxification; glutathione transferase; xenobiotic.

Figures

FIGURE 1
FIGURE 1
Chemical structures of 2,4,6-trinitrotoluene (TNT) and the three glutathione-TNT conjugates, as determined by Gunning et al. (2014).
FIGURE 2
FIGURE 2
Structures of GSTU25 and target residues. (A) Structure of the GSTU25 dimer, with monomers in blue and brown. Oxidized glutathione can be observed in each of the monomer active sites, in stick format. (B) Active site of the GSTU25 monomer showing binding of GSSG. The electron density corresponds to the Fo-Fc omit map contoured at a level of 3σ, and is that which was obtained prior to refinement of the ligand atoms, which have been added from the refined ligand complex for clarity. Side chains of residues conserved between U24 and U25 are shown with side-chain carbon atoms in Blue; Side-chains of residue positions chosen for mutation are shown with side-chain carbon atoms in gold.
FIGURE 3
FIGURE 3
Comparison of GSTU25 with CoGRX2. (A) Superimposed structures of the glutaredoxin subunit from Clostridium oremlandii (CoGRX2 in complex with GSSG (C-atoms in gray), and the GSTU25 subunit (green) in complex with GSSG (C-atoms in green). The RMS value for the superimposed structures is 2.3 Å over 73 residues. (B) Position of the active residue for GSH thiol stabilization: serine 13, in GSTU25 and cysteine 12 in CoGRX2.
FIGURE 4
FIGURE 4
Multiple sequence alignment of Tau class GSTs. Figure generated using Clustal Omega (Sievers et al., 2011).
FIGURE 5
FIGURE 5
TNT-conjugate profiles from GSTs. (A) Total conjugates and (B) conjugate profiles produced by AtGSTU24, AtGSTU25, and mutants. Conjugate 3 = 2-glutathionyl-4,6-dinitrotoluene (GDNT). Results are means of three replicates ± SE, a, significantly different from AtGSTU24, b, significantly different from AtGSTU25.
FIGURE 6
FIGURE 6
Fluorescence-emission spectra of 1-anilino-8-naphthalene-sulfonate (ANS) binding to the active site of the GSTs. (A) Spectra from GSTU24 and GSTU25. (B) Spectra from GSTU24 and its respective mutants. (C) Spectra from GSTU25 and its respective mutants. ANS, blank sample without enzyme; A-I, GSTU24 and GSTU25 mutants as presented in Table 3. Results are means of three technical replicates.
FIGURE 7
FIGURE 7
GST activity using 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) substrate for GSTU24, GSTU25 and their respective mutants. Results are means of three technical replicates ± SE, a, significantly different from AtGSTU24; b, significantly different from AtGSTU25.
FIGURE 8
FIGURE 8
Michaelis–Menten plots from purified GST proteins. (A) GSTU24, (B) ABDCE mutant, and (C) GSTU25, assayed with 1-chloro-2,4- dinitrobenzene (CDNB) substrate. Values represent the mean of at least four reactions ± SE.

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