Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B

Abstract

Widespread contamination of land and groundwater has resulted from the use, manufacture, and storage of the military explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX). This contamination has led to a requirement for a sustainable, low-cost method to remediate this problem. Here, we present the characterization of an unusual microbial P450 system able to degrade RDX, consisting of flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA. The affinity of XplA for the xenobiotic compound RDX is high (K_d = 58 μM) and comparable with the K_m of other P450s toward their natural substrates (ranging from 1 to 500 μM). The maximum turnover (k_cat) is 4.44 per s, only 10-fold less than the fastest self-sufficient P450 reported, BM3. Interestingly, the presence of oxygen determines the final products of RDX degradation, demonstrating that the degradation chemistry is flexible, but both pathways result in ring cleavage and release of nitrite. Carbon monoxide inhibition is weak and yet the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a potent inhibitor. To test the efficacy of this system for the remediation of groundwater, transgenic Arabidopsis plants expressing both xplA and xplB were generated. They are able to remove saturating levels of RDX from liquid culture and soil leachate at rates significantly faster than those of untransformed plants and xplA-only transgenic lines, demonstrating the applicability of this system for the phytoremediation of RDX-contaminated sites.

Cytochrome P450s catalyze a diverse range of chemical reactions including hydroxylation, epoxidation, demethylation, dehalogenation, desaturation, and isomerization (1). As a consequence, P450s are involved in a host of metabolic pathways. In eukaryotes and prokaryotes, they catalyze critical steps in the biosynthesis of key metabolites such as steroids and vitamins (2), fatty acids (3), and lignin (4). P450s have also been established as detoxification enzymes with activity toward a range of xenobiotics. It was thus in keeping with this role that xplA, which was isolated from Rhodococcus rhodochrous 11Y by growth on the explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX) as sole nitrogen source, was found to encode a P450 (5). The synthetic N-NO₂ bond of the RDX molecule, which is rare in nature, was accommodated by this enzyme. Further analysis revealed an unusual arrangement of subunits that contribute to the different steps in P450 catalysis.

Cytochrome P450s, as heme-containing enzymes, require reduction of the heme to activate the catalytic center, a process involving supply of electrons from NAD(P)H to the P450 via partnering enzymes. The nature of the redox partners varies. Class one P450s include bacterial and mitochondrial enzymes that use an FAD-containing ferredoxin reductase-like protein and an iron-sulfur ferredoxin-like protein. Class two P450s are usually bound to the endoplasmic reticulum membrane along with the partnering enzyme, NADPH cytochrome P450 reductase, which contains FAD and FMN domains. Although the P450s and redox partners predominantly exist as separate polypeptides, examples have come to light where the three catalytic domains required for activity are fused together, e.g., the Bacillal BM3 (6), Rhodococcal RhF (7, 8), and the fungal CYP505A1 (8). In all classes, despite the variety of forms, NADPH is usually the source of the electrons, and three electron transfer domains are involved. With XplA, a different arrangement of subunits is seen, with the second electron transfer step, a flavodoxin domain, fused to the P450 domain (9). The organization of the domains is also unusual, with the flavodoxin domain fused to the N terminus of the P450. The first electron transfer step has been postulated to be encoded by a reductase, xplB, adjacent to xplA in the R. rhodochrous genome. XplB has homology to adrenodoxin reductase (5), which transfers electrons from NADPH to adrenodoxin in a synthetically fused P450 (10), and also transfers electrons to flavodoxin (11).

Interest in XplA and XplB has arisen after the contamination of land and groundwater with RDX as a result of the widespread manufacture, use, and disposal of munitions. This contamination is of concern as RDX is toxic to all classes of organisms tested, and the Environmental Protection Agency (EPA) classifies RDX as a priority pollutant. Contamination on military training ranges is of particular concern. For example, the use of RDX has been restricted by the EPA at the Massachusetts Military Reservation of Cape Cod where RDX contamination is threatening drinking water sources (12).

Microorganisms present in soil heavily contaminated with explosives have been found to degrade RDX, but do not possess sufficient biomass or metabolic activity to degrade this compound before it leaches through soils polluting groundwater. Interestingly, to date, xplA and xplB have been found only in Rhodococcus and related bacteria isolated from RDX-contaminated soil, suggesting that the RDX-degrading ability of XplA may have evolved under this selective pressure. XplA has been recombinantly expressed and shown to degrade RDX in vitro with a surrogate reductase (9). In this article, the activity of XplA with its native reductase XplB shows the ability of the proteins to work as efficient partners to degrade RDX. Further characterization is undertaken along with a detailed analysis of the RDX breakdown pathway under anaerobic and aerobic conditions. We have previously demonstrated that expression of XplA in Arabidopsis confers both the ability to remove RDX from liquid culture and resistance to the phytotoxic effects of RDX; however, this activity relies on support from endogenous plant reductases (9). Here, the expression of both xplA and xplB in Arabidopsis enabled the rapid removal of RDX from liquid culture and soil leachate, a rate significantly faster than for plants expressing xplA alone. These results demonstrate that this technology can be applied to remediate RDX from contaminated sites.

Results and Discussion

Optimizing Expression and Assay Conditions.

The purification of XplA to homogeneity has been described (9). Soluble expression and purification of XplB was achieved by using a pGEX vector where GST is fused to the N terminus of XplB (Fig. 1A). Cleavage of the GST resulted in the loss of the 72-kDa XplB band from SDS/PAGE gels, the appearance of several smaller bands, and <5% protein recovery, suggesting insolubility of any cleaved protein (data not shown). The soluble, fused XplB was able to transfer electrons to XplA for the degradation of RDX, whereas GST alone had no such activity (Fig. 1B), therefore fused GST-XplB was used in subsequent studies. Fig. 1C shows that a 2-fold molar excess of XplB to XplA was the ratio at which XplA became limiting (as measured by flavin levels). Conversely, a 10-fold molar excess of XplA to XplB was the ratio at which XplB became limiting (Fig. 1D). At lower ratios, the concentration of both enzymes influenced the rate of activity toward RDX, as expected for a second-order reaction, suggesting that collision of the two subunits is a major rate-limiting contribution. The optimal pH for RDX degradation was pH 6.5–7.0 irrespective of the buffer used, and potassium phosphate at pH 6.8 was used subsequently. Activity was not significantly affected by ionic strength between 0 and 100 mM NaCl, but above 100 mM, an inhibitory effect of sodium chloride was seen (data not shown), thus NaCl was omitted from further assays.

Fig. 1.

Recombinant expression of XplB and assay of activity with XplA. (A) Protein purification on 10% SDS/PAGE. Lane 1, molecular weight markers; lane 2, solubilized recombinant protein; lane 3, affinity-purified XplB. (B) Aerobic activity of GST-XplB (0.26 μM; filled symbol) and GST (0.71 μM; open symbol) with XplA (0.27 μM). (C) Anaerobic activity of 60 nM XplA with various ratios of XplB. (D) Anaerobic activity of 60 nM XplB with various ratios of XplA. Values are the mean ± SD of triplicates.

Spectral Analysis of XplA and XplB.

XplA had previously been shown to contain a classic P450 heme and to be able to bind carbon monoxide when reduced, producing a spectral shift to 450 nm, and a flavin binding domain (9), but the nature of the flavin was not determined. Purified XplB also possessed a classic flavin absorbance spectra, and release of the flavin from XplA and XplB by boiling and subsequent analysis by HPLC showed XplA to contain predominantly FMN and XplB FAD [supporting information (SI) Fig. 8].

Reduction of XplA by sodium dithionite causes a characteristic decrease in the heme 420-nm peak and a shift to a maximum of 389 nm. The dominating heme spectrum masked any flavin absorbance. Six nanomoles of XplA was fully reduced by between 8 and 10 nmol of sodium dithionite, suggesting the majority of XplA has flavin bound (full reduction of heme and flavin would take 9 nmol) (Fig. 2 A and B).

Fig. 2.

Spectral analysis of the reduction of XplA and XplB. (A) UV-visible spectra of 6 nmol of XplA (determined by protein concentration), titrated with the indicated amount of sodium dithionite (nmol) under anaerobic conditions. (B) Difference spectra of the sodium dithionite titration of XplA, generated by subtraction of the original XplA spectrum from those with sodium dithionite. (C) Anaerobic titration of 37 nmol XplB (by protein) with the indicated amount of NADPH (nmol).

On reduction of XplB by NADPH, the flavin absorbance was completely bleached. No changes were observed between 550 and 600 nm, consistent with a lack of the stabilized semiquinone form, and implying a two-electron reduction (Fig. 2C). For XplB, the concentration of NADPH required for full reduction was approximately half the protein concentration, indicating that half of the XplB has flavin bound. Addition of flavin during purification and varying growth conditions did not improve this level. XplB was not readily reduced by NADH nor was NADH a successful electron donor for RDX degradation (data not shown).

RDX Binding and Activity of XplA and XplB.

The binding of RDX to XplA has been examined in two ways. Titration of XplA with RDX in an anaerobic environment revealed the low spin to high spin change in the heme often seen on substrate binding and a binding affinity (K_d) of 57.9 ± 2.8 μM for RDX (Fig. 3 A and B). The K_m was calculated to be 83.7 ± 17.8 μM, and the maximum turnover (k_cat) of the enzyme was 4.44 ± 0.46 per s using a saturating ratio of XplB (2-fold molar excess) and anaerobic conditions (Fig. 3C). The K_m and K_d values are similar to each other, and a turnover of 4.44 per s is comparable with the Pseudomonal P450cam toward its natural substrate camphor (27 per s) (13), perhaps surprising given the xenobiotic nature of RDX. The number of electron transfer steps involved in a P450 system often prevent the turnover from being substantially faster (1).

Fig. 3.

XplA and RDX binding. All analyses were carried out anaerobically. (A) UV-visible spectral changes in 4.5 nmol XplA on the addition of 0–150 nmol of RDX (in DMSO). (Inset) Difference spectra generated by subtraction of nonbound XplA spectrum from the spectra of XplA with RDX. (B) Plot of A₃₉₁ − A₄₂₅ generated from the difference spectra against RDX concentration. (C) Initial rates of substrate use at a range of RDX concentrations plotted against RDX concentration with 60 nM XplA and 120 nM XplB. Values are the mean ± SD of triplicates.

RDX Breakdown Pathways.

Initially, the breakdown products of RDX degradation were analyzed anaerobically to determine the amount of NADPH required without losses to uncoupled cleavage of oxygen. Formaldehyde and nitrite were measured directly, whereas RDX and other products were assayed after freezing the samples. It was surprising to find ratios of nitrite to RDX, after 70 min, of 1.4:1.0 and formaldehyde to RDX of 1.96:1.00 as it was previously thought that the breakdown pathway would follow that proposed by Fournier et al. (14) with 2:1 nitrite and 1:1 formaldehyde and production of 4-nitro-2,4, diazabutanal (NDAB) (Fig. 4A). NDAB was not detected; however, the RDX breakdown product methylenedinitramine (MEDINA) was, reaching a ratio of 0.68:1 after 70 min and then decreasing, possibly because of instability in water (15). The ratio of NADPH to RDX degraded was 1.26:1.00 at 70 min, suggesting these compounds are tightly coupled.

Fig. 4.

Mass balance of RDX breakdown. (A) Anaerobic degradation of RDX (60 nM XplA and XplB) and analyses were carried out as in Materials and Methods. Controls with boiled XplA and XplB (not shown) contained the following levels of analytes (nmol/ml) over the time course: RDX, 100 ± 4.3; nitrite, 0 ± 3.9; formaldehyde, 0 ± 6.3; and MEDINA, 0–6.3. (B) Aerobic degradation of RDX. Reactions contained 90 nM XplA and XplB. Controls with boiled XplA and XplB (not shown) contained RDX (100 ± 2.5), nitrite (0 ± 2), formaldehyde (0 ± 16), and NDAB (0–2.6) over the time course. Values are the mean ± SD of triplicates.

When the breakdown pathway under aerobic conditions was examined, a different picture arose with an increase in the ratio of nitrite to RDX to 2.49:1.00 after 130 min and a decrease in the formaldehyde to RDX ratio to 1.4:1.0 (Fig. 4B). NDAB was detected, and levels continued to increase throughout the time course; MEDINA was not detected.

The mechanism for denitration of RDX by XplA is not yet fully known; however, once denitration and hydration have occurred, whether under aerobic or anaerobic conditions, the resulting imine intermediate would be highly unstable in water and spontaneously decompose (16). Under anaerobic conditions, mono-denitration and mono-hydration of RDX followed by ring cleavage would produce MEDINA (Fig. 5, route A). Under aerobic conditions, it is proposed that RDX is subjected to di-denitration–di-hydration before ring cleavage. This mechanism, leading to the formation of NDAB (see Fig. 5, route B), has been described (14). Degradation of RDX with radiolabeled oxygen and another P450 system suggests that direct hydroxylation of the RDX molecule by XplA is unlikely (17). The role oxygen plays in the mechanism of RDX degradation by XplA, either within the enzyme or within the reaction environment, is therefore currently unclear. The pathway presented in Fig. 5 is based on the analysis of the final degradation products and the proposed intermediates by analogy from previously published work (14, 17). Additional work involving labeled RDX and deuterated solvent may allow precise details of the mechanism to be determined.

Fig. 5.

Proposed degradation pathway of RDX under aerobic and anaerobic conditions. Ring cleavage occurs at ab under anaerobic conditions (route A) and at cb under aerobic conditions (route B). Compounds in brackets are hypothetical, and the mechanisms are based on detection of nitrite and formaldehyde, the final products, and analogy with previous work (14, 17).

Other examples of P450s catalyzing different reactions depending on the presence of oxygen are known. For example, under anaerobic conditions human CYP2A6 and CYP101 both catalyze reductive reactions toward halogenated substrates, whereas under aerobic conditions, CYP2A6 catalyzes dehalogenation, (18) and CYP101 catalyzes a hydroxylation reaction (19).

XplA Activity with a Wider Range of Substrates and Inhibitors.

RDX as a synthetic compound may not be the native substrate for XplA, so the activity toward a range of other substrates was tested. The related nitramine explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), was not transformed at the aqueous solubility limit for HMX of 15 μM, even after several hours of incubation. Trace levels of XplA activity toward the nitroaromatic explosive trinitrotoluene (TNT) were found (turnover was <1% per h at 100 μM), but transformation products could not be identified. The heme spectrum of XplA was not altered by the presence of TNT. To test the ability of XplA to perform classic P450 hydroxylation and demethylation reactions, established P450 substrates were tested. No hydroxylating activity was detected toward testosterone or paclitaxel, nor demethylating activity toward 7-ethoxycoumarin or ethoxyresorufin. However, oxidizing activity was detected toward both methyl tolyl and methyl phenyl sulfides generating sulfoxide products (data not shown).

Methyl tolyl sulfide was also shown to inhibit RDX catabolism, as was the P450-specific inhibitor metyrapone. The strongest inhibition was seen by TNT (Fig. 6), whereas the rate of carbon monoxide inhibition was less than expected, given the usual high affinity of P450s for carbon monoxide. This inhibition was not increased by a higher concentration of carbon monoxide. XplA may have a lower affinity for CO in the presence of RDX, as seen with other substrates of P450s (20).

Fig. 6.

Activity of XplA and XplB with inhibitors. Standard anaerobic activity assays using 100 μM RDX as substrate were carried out in the presence of 100 μM metyrapone or 100 μM TNT, and activity was compared with that with only RDX (100%). Carbon monoxide (CO) and methyl tolyl sulfide (MTS) inhibition was tested in standard aerobic conditions using 100 μM RDX as substrate. Values are the mean ± SD of triplicates.

Application of the Enzymes for Phytodegradation of RDX.

It has previously been shown that transgenic plants expressing xplA can degrade RDX (9), but this activity relies on the availability of endogenous plant reductases, which may be limiting. Thus, xplB was transformed into Arabidopsis, and transgenic plant lines expressing both xplA and xplB were generated. A previously characterized plant line expressing xplA (XplA-10) (9) was used to produce five independently transformed lines expressing xplB. In addition, five independently transformed lines expressing only xplB were characterized. The results presented here are from plants homozygous for these transgenes. Quantitative analysis by real-time PCR showed that xplA was expressed in all five XplAB lines (Fig. 7A), although expression levels varied from that of the original parental line, XplA-10. A range in the level of xplB transcript was seen; with line XplAB-27 exhibiting the highest levels of both xplA and xplB transcripts (Fig. 7A). These lines were grown in axenic liquid culture to determine rates of RDX uptake. As reported (9), line XplA-10 removed all 180 μM RDX from the medium within 5 days; however, the XplAB lines removed the RDX significantly faster, with lines XplAB-2 and XplAB-27 removing >50% of the RDX within 4 h, 30 times faster than the XplA-10 line (Fig. 7B). The xplB-only lines had uptake rates similar to those of untransformed, wild-type plants (Fig. 7C). NDAB and MEDINA levels were not measured in the liquid culture or soil-grown plants. Liquid culture-grown plants and water-saturated soil-grown roots are likely to be hypoxic. It is possible from our characterization that, depending on oxygen availability, either NDAB or MEDINA is produced by xplA–expressing plants.

Fig. 7.

Characterization of XplAB and XplB transgenic Arabidopsis lines. (A) Expression of xplA and xplB in rosette leaves. Quantitative analysis was done by real-time PCR of xplA and xplB transcript abundance in rosette leaves of the transgenic lines relative to line XplAB-2. ACTIN2 mRNA was used as an internal reference. Results represent the mean of three independent RNA isolations measured in duplicate from the pooled rosette leaves of 10 plants ± SE. (B and C) Uptake of RDX from media by Arabidopsis seedlings. Results are the mean ± SE of five replicate flasks, each containing 200 10-day-old seedlings. (D) Levels of RDX in soil leachate from Arabidopsis plants watered with 180 μM RDX. Results are the mean ± SE of five replicate pots. NPC, no plant control.

To investigate the ability of the XplAB lines to reduce levels of RDX in contaminated ground water, 8-week-old plants were watered with 180 μM RDX. After 1 week, the soil was flushed with water and the level of RDX in the soil leachate was measured. After this time, the level of RDX in the leachate from untransformed, wild-type plants was unaltered, whereas leachate from the XplA-10 line had decreased by 25%. The RDX in the leachate from lines XplAB-2 and XplAB27 had decreased by 90–97% (Fig. 7D).

Conclusions

XplA and XplB constitute a novel P450 redox system and together efficiently degrade the xenobiotic RDX. Degradation follows two different routes dependent on the presence of oxygen. One mole of MEDINA and nitrite are the dominant products anaerobically, whileas 1 mol of NDAB and 2 mol of nitrite are produced in aerobic conditions. With both pathways resulting in ring cleavage and nitrite release, applications of these enzymes for bioremediation look encouraging. One of the biggest concerns of RDX as a pollutant is that it migrates readily through soil into the groundwater and subsequently contaminates drinking water supplies. Here, we show that Arabidopsis plants expressing xplA and xplB have the ability to effectively remove RDX from the soil leachate. The studies here illustrate that these genes, or possibly an xplA–xplB gene fusion, could be engineered into plant species suited to growth on military training ranges and used to remediate RDX.

Materials and Methods

RDX was supplied by the Defense Science and Technology Laboratory at the U.K. Ministry of Defense (Fort Halstead, Kent, U.K.). MEDINA and NDAB were provided by Ron Spanggord (SRI International, Menlo Park, CA).

XplA and XplB Expression and Purification.

The xplA gene was cloned and expressed as described (9). The xplB gene was amplified from pHSX1 by PCR using primers containing overhanging BamH1 sites, 5′-GGATCCGACATCATGAGTGAAGTGGAC and 3′-GGATCCGCAGACCGATTCGGCCGG and ligated into pGEX2T (Merck Chemicals, Nottingham, U.K.), which engineers an N-terminal GST domain. The construct was sequenced, transformed into Escherichia coli BL21 (DE3) and grown at 20°C in Luria broth containing 100 μg/ml carbenicillin to OD₆₀₀ (1.0), 1 mM isopropyl β-d-thiogalactopyranoside was added, and the culture was grown for an additional 24 h. The cell pellet was resuspended in 10 ml of PBS (140 mM NaCl/15 mM KH₂PO₄/80 mM Na₂HPO₄) containing 0.2 mM PMSF and lysed at 1,500 psi (1 psi = 6.89 kPa) in a French Press (Thermo IEC). The lysate was centrifuged (10,000 × g for 15 min), and the soluble protein was purified by using 200 μl of 50% glutathione-coupled Sepharose gel (Amersham, Piscataway, NJ) according to the manufacturer's instructions and recovered in glutathione elution buffer (20 mM reduced glutathione/100 mM Tris·HCl, pH 8.5/120 mM NaCl). Protein assays were carried out with Coomassie Protein Assay Reagent (Pierce, Rockford, IL) using BSA as reference. Proteins were analyzed as described (21).

Flavin Content Determination.

The flavin content of XplA and XplB was determined after boiling for 20 min then centrifugation of the precipitated protein and trichloroacetic acid precipitation (10% of 240 mg/ml), followed by centrifugation and sodium bicarbonate neutralization. Both methods gave similar results for each protein. The concentration of released flavin was used to measure XplA and XplB concentration for activity assays. The released flavins were identified by HPLC according to ref. 22 and compared with commercially available standards for identification.

Spectral Analyses.

Spectral analyses were performed on a spectrophotometer (v560; Jasco, Easton, MD) scanning between 800 and 300 nm at 400 nm/min. Anaerobic analyses were carried out in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI). For all spectral analyses, enzyme concentrations quoted are for sample protein. The XplA titration with sodium dithionite in the anaerobic chamber followed desalting of the protein on a PD-10 column (GE Healthcare) into 50 mM potassium phosphate, pH 6.8 (bubbled with nitrogen before equilibrating in the chamber for 2 days). For RDX titration, RDX was dissolved in DMSO and no more than 3 μl was added per ml.

XplA and XplB Activity Assays Toward RDX.

Anaerobic conditions.

XplA and XplB were placed on ice in an anaerobic chamber in open vials for 3 h for the palladium catalyst to remove oxygen. Buffer was bubbled with nitrogen before equilibration in the chamber. The reaction mixture contained 60 nM XplA and XplB in 50 mM potassium phosphate (pH 6.8), 300 μM NADPH, and 100 μM RDX in a total volume of 1 ml. Other reagents and solutions were placed overnight in the anaerobic chamber with a nitrogen atmosphere, before use. Oxygen levels were monitored with a model 10 gas analyzer (Coy Laboratory Products) and maintained <1 ppm by using a 5% (vol/vol) hydrogen mix in the presence of a palladium catalyst.

Aerobic conditions.

The reaction mixture contained 175 nM XplA and 150 nM XplB, 50 mM potassium phosphate (pH 6.8), 300 μM NADPH, 100 μM RDX, 0.72 units Thermoanaerobium brockii alcohol dehydrogenase (Sigma, St. Louis, MO), and 30 μl isopropanol in a total volume of 1 ml. All reactions were performed at room temperature (20°C), and assays were stopped by the addition of 10% trichloroacetic acid (240 mg/ml) or, for the mass balance experiments, 30-kDa cut-off spin columns (Microcon YM-30; Amicon). The reaction was initiated by the addition of RDX.

Analysis of Products.

RDX removal was measured by using RP-HPLC with a HPLC system (2695 Separations Module and 2996 Photodiode Array Detector; Waters) using a Techsphere C18 column (250 × 4.6 mm) under isocratic conditions of 60% water and 40% acetonitrile at a flow rate of 1 ml/min over 10 min. RDX elution was monitored at 205 nm, and intergrations were performed with Empower software.

Formaldehyde analysis was carried out according to Nash (23). Nitrite analysis followed the method of Scheideler and Ninnemann (24) terminated by removal of proteins with 30-kDa cut-off spin columns. The concentration of NDAB and MEDINA was determined by using an HPLC system as reported (25).

Activity Assays Toward Other Substrates and with Inhibitors.

Analysis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine was as described for RDX. Loss of TNT was analyzed by using a water mobile phase of 50% MeCN, 50% water over 12 min with monitoring at 230 nm. Paclitaxel was analyzed by using a methanol and water gradient from 60–70% MeOH over 20 min with monitoring at 230 nm. Testosterone was analyzed by HPLC using a 58–62% MeOH gradient over 8 min. Activity toward 7-ethoxycoumarin was tested by following fluorescence of 7-hydroxy coumarin, with excitation at 350 nm and emission at 450 nm adapted from ref. 26 and TLC. Activity toward ethoxyresorufin was determined fluorometrically with excitation at 530 nm and emission at 590 nm, adapted from ref. 26. Carbon monoxide inhibition was carried out aerobically by adding 100 or 500 μl per ml of carbon monoxide-saturated buffer.

Plant Transformation Methods.

The xplB gene was cloned into the binary vector pART27 (27) under the control of the CaMV35S promoter and ocs terminator, and transformed by Agrobacterium-mediated floral dipping into wild-type and xplA-expressing Arabidopsis thaliana, ecotype Columbia-0 as in ref. 9.

Liquid Culture and Soil Leachate Experiments.

Liquid culture experiments were performed as described (9). Soil leachate studies were carried out on 6-week-old Arabidopsis plants grown under 180 μm·m²·s ⁻¹ light in a 12-h photoperiod. Plants were grown in pots containing 30 g of uncontaminated soil (Levingtons F2 compost), five plants per pot. Each pot was flooded with 50 ml of 180 μM RDX, then 7 days later, flushed through with 50 ml of water. The collected soil leachates were analyzed for RDX content by using HPLC as described above.

Abbreviations

RDX

hexa-hydro-1,3,5-trinitro-1,3,5-triazine

TNT

trinitrotoluene

NDAB

4-nitro-2,4, diazabutanal

MEDINA

methylenedinitramine.