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

Rosamond G. Jackson*, Elizabeth L. Rylott*, Diane Fournier†, Jalal Hawari†, and Neil C. Bruce*‡

*Center for Novel Agricultural Products, Department of Biology, University of York, P.O. Box 373, York YO10 5YW, ; and †Biotechnology Research Institute, National Research Council of , Montreal, QC, Canada H4P 2R2

Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved September 10, 2007 (received for review May 31, 2007) Widespread contamination of land and groundwater has resulted P450s and redox partners predominantly exist as separate from the use, manufacture, and storage of the military explosive polypeptides, examples have come to light where the three hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX). This contamination catalytic domains required for activity are fused together, e.g., has led to a requirement for a sustainable, low-cost method to the Bacillal BM3 (6), Rhodococcal RhF (7, 8), and the fungal remediate this problem. Here, we present the characterization of CYP505A1 (8). In all classes, despite the variety of forms, an unusual microbial P450 system able to degrade RDX, consisting NADPH is usually the source of the electrons, and three electron of flavodoxin reductase XplB and fused flavodoxin-cytochrome transfer domains are involved. With XplA, a different arrange- P450 XplA. The affinity of XplA for the xenobiotic compound RDX ment of subunits is seen, with the second electron transfer step, ␮ ؍ is high (Kd 58 M) and comparable with the Km of other P450s a flavodoxin domain, fused to the P450 domain (9). The toward their natural substrates (ranging from 1 to 500 ␮M). The organization of the domains is also unusual, with the flavodoxin maximum turnover (kcat) is 4.44 per s, only 10-fold less than the domain fused to the N terminus of the P450. The first electron fastest self-sufficient P450 reported, BM3. Interestingly, the pres- transfer step has been postulated to be encoded by a reductase, ence of oxygen determines the final products of RDX degradation, xplB, adjacent to xplA in the R. rhodochrous genome. XplB has demonstrating that the degradation chemistry is flexible, but both homology to adrenodoxin reductase (5), which transfers elec- pathways result in ring cleavage and release of nitrite. Carbon trons from NADPH to adrenodoxin in a synthetically fused P450 monoxide inhibition is weak and yet the nitroaromatic explosive (10), and also transfers electrons to flavodoxin (11). 2,4,6-trinitrotoluene (TNT) is a potent inhibitor. To test the efficacy Interest in XplA and XplB has arisen after the contamination of this system for the remediation of groundwater, transgenic of land and groundwater with RDX as a result of the widespread Arabidopsis plants expressing both xplA and xplB were generated. manufacture, use, and disposal of munitions. This contamination They are able to remove saturating levels of RDX from liquid is of concern as RDX is toxic to all classes of organisms tested, culture and soil leachate at rates significantly faster than those of and the Environmental Protection Agency (EPA) classifies untransformed plants and xplA-only transgenic lines, demonstrat- RDX as a priority pollutant. Contamination on military training ing the applicability of this system for the of ranges is of particular concern. For example, the use of RDX has RDX-contaminated sites. been restricted by the EPA at the Massachusetts Military Reservation of Cape Cod where RDX contamination is threat- cytochrome P450 ͉ phytoremediation ͉ hexa-hydro-1,3,5-trinitro- ening drinking water sources (12). 1,3,5-triazine Microorganisms present in soil heavily contaminated with ex- plosives have been found to degrade RDX, but do not possess ytochrome P450s catalyze a diverse range of chemical reac- sufficient biomass or metabolic activity to degrade this compound Ctions including hydroxylation, epoxidation, demethylation, before it leaches through soils polluting groundwater. Interestingly, dehalogenation, desaturation, and isomerization (1). As a con- to date, xplA and xplB have been found only in Rhodococcus and sequence, P450s are involved in a host of metabolic pathways. In related bacteria isolated from RDX-contaminated soil, suggesting eukaryotes and prokaryotes, they catalyze critical steps in the that the RDX-degrading ability of XplA may have evolved under biosynthesis of key metabolites such as steroids and vitamins (2), this selective pressure. XplA has been recombinantly expressed and fatty acids (3), and lignin (4). P450s have also been established shown to degrade RDX in vitro with a surrogate reductase (9). In as detoxification enzymes with activity toward a range of xeno- this article, the activity of XplA with its native reductase XplB biotics. It was thus in keeping with this role that xplA, which was shows the ability of the proteins to work as efficient partners to isolated from Rhodococcus rhodochrous 11Y by growth on the degrade RDX. Further characterization is undertaken along with explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX) as sole a detailed analysis of the RDX breakdown pathway under anaer- nitrogen source, was found to encode a P450 (5). The synthetic obic and aerobic conditions. We have previously demonstrated that N-NO bond of the RDX molecule, which is rare in nature, was 2 expression of XplA in Arabidopsis confers both the ability to remove accommodated by this enzyme. Further analysis revealed an unusual arrangement of subunits that contribute to the different steps in P450 catalysis. Author contributions: R.G.J., E.L.R., J.H., and N.C.B. designed research; R.G.J., E.L.R., and Cytochrome P450s, as heme-containing enzymes, require D.F. performed research; R.G.J., E.L.R., D.F., J.H., and N.C.B. analyzed data; and R.G.J., E.L.R., reduction of the heme to activate the catalytic center, a process J.H., and N.C.B. wrote the paper. involving supply of electrons from NAD(P)H to the P450 via The authors declare no conflict of interest. partnering enzymes. The nature of the redox partners varies. This article is a PNAS Direct Submission. Class one P450s include bacterial and mitochondrial enzymes Abbreviations: RDX, hexa-hydro-1,3,5-trinitro-1,3,5-triazine; TNT, trinitrotoluene; NDAB, that use an FAD-containing ferredoxin reductase-like protein 4-nitro-2,4, diazabutanal; MEDINA, methylenedinitramine. and an iron-sulfur ferredoxin-like protein. Class two P450s are ‡To whom correspondence should be addressed. E-mail: [email protected]. usually bound to the endoplasmic reticulum membrane along This article contains supporting information online at www.pnas.org/cgi/content/full/ with the partnering enzyme, NADPH cytochrome P450 reduc- 0705110104/DC1. tase, which contains FAD and FMN domains. Although the © 2007 by The National Academy of Sciences of the USA

16822–16827 ͉ PNAS ͉ October 23, 2007 ͉ vol. 104 ͉ no. 43 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705110104 Downloaded by guest on September 30, 2021 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 ␮M; filled symbol) and GST (0.71 ␮M; open symbol) with XplA (0.27 ␮M). (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 Ϯ SD of triplicates.

RDX from liquid culture and resistance to the phytotoxic effects of 600 nm, consistent with a lack of the stabilized semiquinone form, RDX; however, this activity relies on support from endogenous and implying a two-electron reduction (Fig. 2C). For XplB, the plant reductases (9). Here, the expression of both xplA and xplB in concentration of NADPH required for full reduction was approx- Arabidopsis enabled the rapid removal of RDX from liquid culture and soil leachate, a rate significantly faster than for plants express- ing 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 Ͻ5% 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). Con- versely, 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, suggest-

ing that collision of the two subunits is a major rate-limiting SCIENCES contribution. The optimal pH for RDX degradation was pH

6.5–7.0 irrespective of the buffer used, and potassium phosphate APPLIED BIOLOGICAL 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.

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 character- istic 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 be- Fig. 2. Spectral analysis of the reduction of XplA and XplB. (A) UV-visible tween 8 and 10 nmol of sodium dithionite, suggesting the spectra of 6 nmol of XplA (determined by protein concentration), titrated with the indicated amount of sodium dithionite (nmol) under anaerobic condi- majority of XplA has flavin bound (full reduction of heme and tions. (B) Difference spectra of the sodium dithionite titration of XplA, gen- flavin would take 9 nmol) (Fig. 2 A and B). erated by subtraction of the original XplA spectrum from those with sodium On reduction of XplB by NADPH, the flavin absorbance was dithionite. (C) Anaerobic titration of 37 nmol XplB (by protein) with the completely bleached. No changes were observed between 550 and indicated amount of NADPH (nmol).

Jackson et al. PNAS ͉ October 23, 2007 ͉ vol. 104 ͉ no. 43 ͉ 16823 Downloaded by guest on September 30, 2021 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 Ϯ 4.3; nitrite, 0 Ϯ 3.9; , 0 Ϯ 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 Ϯ 2.5), nitrite (0 Ϯ 2), formaldehyde (0 Ϯ 16), and NDAB (0–2.6) over the time course. Values are the mean Ϯ 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 cleav- age of oxygen. Formaldehyde and nitrite were measured directly, Fig. 3. XplA and RDX binding. All analyses were carried out anaerobically. whereas RDX and other products were assayed after freezing (A) UV-visible spectral changes in 4.5 nmol XplA on the addition of 0–150 nmol the samples. It was surprising to find ratios of nitrite to RDX, of RDX (in DMSO). (Inset) Difference spectra generated by subtraction of after 70 min, of 1.4:1.0 and formaldehyde to RDX of 1.96:1.00 nonbound XplA spectrum from the spectra of XplA with RDX. (B) Plot of A391 Ϫ as it was previously thought that the breakdown pathway would A425 generated from the difference spectra against RDX concentration. (C) follow that proposed by Fournier et al. (14) with 2:1 nitrite and Initial rates of substrate use at a range of RDX concentrations plotted against 1:1 formaldehyde and production of 4-nitro-2,4, diazabutanal Ϯ RDX concentration with 60 nM XplA and 120 nM XplB. Values are the mean (NDAB) (Fig. 4A). NDAB was not detected; however, the RDX SD of triplicates. breakdown product methylenedinitramine (MEDINA) was, reaching a ratio of 0.68:1 after 70 min and then decreasing, imately half the protein concentration, indicating that half of the possibly because of instability in water (15). The ratio of NADPH XplB has flavin bound. Addition of flavin during purification and to RDX degraded was 1.26:1.00 at 70 min, suggesting these varying growth conditions did not improve this level. XplB was not compounds are tightly coupled. readily reduced by NADH nor was NADH a successful electron When the breakdown pathway under aerobic conditions was donor for RDX degradation (data not shown). 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 RDX Binding and Activity of XplA and XplB. The binding of RDX to the formaldehyde to RDX ratio to 1.4:1.0 (Fig. 4B). NDAB was XplA has been examined in two ways. Titration of XplA with RDX detected, and levels continued to increase throughout the time in an anaerobic environment revealed the low spin to high spin course; MEDINA was not detected. change in the heme often seen on substrate binding and a binding The mechanism for denitration of RDX by XplA is not yet affinity (Kd)of57.9Ϯ 2.8 ␮M for RDX (Fig. 3 A and B). The Km fully known; however, once denitration and hydration have was calculated to be 83.7 Ϯ 17.8 ␮M, and the maximum turnover occurred, whether under aerobic or anaerobic conditions, the (kcat) of the enzyme was 4.44 Ϯ 0.46 per s using a saturating ratio resulting imine intermediate would be highly unstable in water of XplB (2-fold molar excess) and anaerobic conditions (Fig. 3C). and spontaneously decompose (16). Under anaerobic condi- The Km and Kd values are similar to each other, and a turnover of tions, mono-denitration and mono-hydration of RDX followed 4.44 per s is comparable with the Pseudomonal P450cam toward its by ring cleavage would produce MEDINA (Fig. 5, route A). natural substrate camphor (27 per s) (13), perhaps surprising given Under aerobic conditions, it is proposed that RDX is subjected the xenobiotic nature of RDX. The number of electron transfer to di-denitration–di-hydration before ring cleavage. This mech- steps involved in a P450 system often prevent the turnover from anism, leading to the formation of NDAB (see Fig. 5, route B), being substantially faster (1). has been described (14). Degradation of RDX with radiolabeled

16824 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705110104 Jackson et al. Downloaded by guest on September 30, 2021 Fig. 6. Activity of XplA and XplB with inhibitors. Standard anaerobic activity assays using 100 ␮M RDX as substrate were carried out in the presence of 100 ␮M metyrapone or 100 ␮M 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 ␮M RDX as substrate. Values are the mean Ϯ SD of triplicates.

Fig. 5. Proposed degradation pathway of RDX under aerobic and anaerobic increased by a higher concentration of carbon monoxide. XplA conditions. Ring cleavage occurs at ab under anaerobic conditions (route A) may have a lower affinity for CO in the presence of RDX, as seen and at cb under aerobic conditions (route B). Compounds in brackets are with other substrates of P450s (20). hypothetical, and the mechanisms are based on detection of nitrite and formaldehyde, the final products, and analogy with previous work (14, 17). Application of the Enzymes for Phytodegradation of RDX. It has previously been shown that transgenic plants expressing xplA can oxygen and another P450 system suggests that direct hydroxy- degrade RDX (9), but this activity relies on the availability of lation of the RDX molecule by XplA is unlikely (17). The role endogenous plant reductases, which may be limiting. Thus, xplB oxygen plays in the mechanism of RDX degradation by XplA, was transformed into Arabidopsis, and transgenic plant lines either within the enzyme or within the reaction environment, is expressing both xplA and xplB were generated. A previously therefore currently unclear. The pathway presented in Fig. 5 is characterized plant line expressing xplA (XplA-10) (9) was used based on the analysis of the final degradation products and the to produce five independently transformed lines expressing xplB. proposed intermediates by analogy from previously published In addition, five independently transformed lines expressing only work (14, 17). Additional work involving labeled RDX and xplB were characterized. The results presented here are from deuterated solvent may allow precise details of the mechanism plants homozygous for these transgenes. Quantitative analysis by to be determined. real-time PCR showed that xplA was expressed in all five XplAB Other examples of P450s catalyzing different reactions depend- lines (Fig. 7A), although expression levels varied from that of the ing on the presence of oxygen are known. For example, under original parental line, XplA-10. A range in the level of xplB anaerobic conditions human CYP2A6 and CYP101 both catalyze transcript was seen; with line XplAB-27 exhibiting the highest levels of both xplA and xplB transcripts (Fig. 7A). These lines reductive reactions toward halogenated substrates, whereas under were grown in axenic liquid culture to determine rates of RDX aerobic conditions, CYP2A6 catalyzes dehalogenation, (18) and uptake. As reported (9), line XplA-10 removed all 180 ␮MRDX SCIENCES CYP101 catalyzes a hydroxylation reaction (19). from the medium within 5 days; however, the XplAB lines removed the RDX significantly faster, with lines XplAB-2 and APPLIED BIOLOGICAL XplA Activity with a Wider Range of Substrates and Inhibitors. RDX XplAB-27 removing Ͼ50% of the RDX within 4 h, 30 times as a synthetic compound may not be the native substrate for faster than the XplA-10 line (Fig. 7B). The xplB-only lines had XplA, so the activity toward a range of other substrates was uptake rates similar to those of untransformed, wild-type plants tested. The related nitramine explosive, octahydro-1,3,5,7- (Fig. 7C). NDAB and MEDINA levels were not measured in the tetranitro-1,3,5,7-tetrazocine (HMX), was not transformed at ␮ liquid culture or soil-grown plants. Liquid culture-grown plants the aqueous solubility limit for HMX of 15 M, even after and water-saturated soil-grown roots are likely to be hypoxic. It several hours of incubation. Trace levels of XplA activity toward is possible from our characterization that, depending on oxygen the nitroaromatic explosive trinitrotoluene (TNT) were found availability, either NDAB or MEDINA is produced by xplA– Ͻ ␮ (turnover was 1% per h at 100 M), but transformation expressing plants. products could not be identified. The heme spectrum of XplA To investigate the ability of the XplAB lines to reduce levels was not altered by the presence of TNT. To test the ability of of RDX in contaminated ground water, 8-week-old plants were XplA to perform classic P450 hydroxylation and demethylation watered with 180 ␮M RDX. After 1 week, the soil was flushed reactions, established P450 substrates were tested. No hydroxy- with water and the level of RDX in the soil leachate was lating activity was detected toward testosterone or paclitaxel, nor measured. After this time, the level of RDX in the leachate from demethylating activity toward 7-ethoxycoumarin or untransformed, wild-type plants was unaltered, whereas leachate ethoxyresorufin. However, oxidizing activity was detected to- from the XplA-10 line had decreased by 25%. The RDX in the ward both methyl tolyl and methyl phenyl sulfides generating leachate from lines XplAB-2 and XplAB27 had decreased by sulfoxide products (data not shown). 90–97% (Fig. 7D). Methyl tolyl sulfide was also shown to inhibit RDX catabolism, as was the P450-specific inhibitor metyrapone. The strongest Conclusions inhibition was seen by TNT (Fig. 6), whereas the rate of carbon XplA and XplB constitute a novel P450 redox system and monoxide inhibition was less than expected, given the usual high together efficiently degrade the xenobiotic RDX. Degradation affinity of P450s for carbon monoxide. This inhibition was not follows two different routes dependent on the presence of

Jackson et al. PNAS ͉ October 23, 2007 ͉ vol. 104 ͉ no. 43 ͉ 16825 Downloaded by guest on September 30, 2021 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 Ϯ SE. (B and C) Uptake of RDX from media by Arabidopsis seedlings. Results are the mean Ϯ 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 ␮M RDX. Results are the mean Ϯ SE of five replicate pots. NPC, no plant control.

oxygen. One mole of MEDINA and nitrite are the dominant Na2HPO4) containing 0.2 mM PMSF and lysed at 1,500 psi (1 psi ϭ products anaerobically, whileas 1 mol of NDAB and 2 mol of 6.89 kPa) in a French Press (Thermo IEC). The lysate was nitrite are produced in aerobic conditions. With both pathways centrifuged (10,000 ϫ g for 15 min), and the soluble protein was resulting in ring cleavage and nitrite release, applications of purified by using 200 ␮l of 50% glutathione-coupled Sepharose gel these enzymes for look encouraging. One of the (Amersham, Piscataway, NJ) according to the manufacturer’s biggest concerns of RDX as a pollutant is that it migrates readily instructions and recovered in glutathione elution buffer (20 mM through soil into the groundwater and subsequently contami- reduced glutathione/100 mM Tris⅐HCl, pH 8.5/120 mM NaCl). nates drinking water supplies. Here, we show that Arabidopsis Protein assays were carried out with Coomassie Protein Assay plants expressing xplA and xplB have the ability to effectively Reagent (Pierce, Rockford, IL) using BSA as reference. Proteins remove RDX from the soil leachate. The studies here illustrate were analyzed as described (21). that these genes, or possibly an xplA–xplB gene fusion, could be engineered into plant species suited to growth on military Flavin Content Determination. The flavin content of XplA and XplB training ranges and used to remediate RDX. was determined after boiling for 20 min then centrifugation of the precipitated protein and trichloroacetic acid precipitation (10% of Materials and Methods 240 mg/ml), followed by centrifugation and sodium bicarbonate RDX was supplied by the Defense Science and Technology neutralization. Both methods gave similar results for each protein. Laboratory at the U.K. Ministry of Defense (Fort Halstead, The concentration of released flavin was used to measure XplA and Kent, U.K.). MEDINA and NDAB were provided by Ron XplB concentration for activity assays. The released flavins were Spanggord (SRI International, Menlo Park, CA). identified by HPLC according to ref. 22 and compared with commercially available standards for identification. XplA and XplB Expression and Purification. The xplA gene was cloned and expressed as described (9). The xplB gene was amplified from Spectral Analyses. Spectral analyses were performed on a spec- pHSX1 by PCR using primers containing overhanging BamH1 trophotometer (v560; Jasco, Easton, MD) scanning between 800 sites, 5Ј-GGATCCGACATCATGAGTGAAGTGGAC and 3Ј- and 300 nm at 400 nm/min. Anaerobic analyses were carried out GGATCCGCAGACCGATTCGGCCGG and ligated into in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, pGEX2T (Merck Chemicals, Nottingham, U.K.), which engineers MI). For all spectral analyses, enzyme concentrations quoted are an N-terminal GST domain. The construct was sequenced, trans- for sample protein. The XplA titration with sodium dithionite in formed into Escherichia coli BL21 (DE3) and grown at 20°C in the anaerobic chamber followed desalting of the protein on a Luria broth containing 100 ␮g/ml carbenicillin to OD600 (1.0), 1 PD-10 column (GE Healthcare) into 50 mM potassium phos- mM isopropyl ␤-d-thiogalactopyranoside was added, and the cul- phate, pH 6.8 (bubbled with nitrogen before equilibrating in the ture was grown for an additional 24 h. The cell pellet was resus- chamber for 2 days). For RDX titration, RDX was dissolved in pended in 10 ml of PBS (140 mM NaCl/15 mM KH2PO4/80 mM DMSO and no more than 3 ␮l was added per ml.

16826 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705110104 Jackson et al. Downloaded by guest on September 30, 2021 XplA and XplB Activity Assays Toward RDX. Anaerobic conditions. XplA Activity Assays Toward Other Substrates and with Inhibitors. Analysis and XplB were placed on ice in an anaerobic chamber in open of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine was as de- vials for 3 h for the palladium catalyst to remove oxygen. Buffer scribed for RDX. Loss of TNT was analyzed by using a water was bubbled with nitrogen before equilibration in the chamber. mobile phase of 50% MeCN, 50% water over 12 min with The reaction mixture contained 60 nM XplA and XplB in 50 mM monitoring at 230 nm. Paclitaxel was analyzed by using a potassium phosphate (pH 6.8), 300 ␮M NADPH, and 100 ␮M methanol and water gradient from 60–70% MeOH over 20 min RDX in a total volume of 1 ml. Other reagents and solutions with monitoring at 230 nm. Testosterone was analyzed by HPLC were placed overnight in the anaerobic chamber with a nitrogen using a 58–62% MeOH gradient over 8 min. Activity toward atmosphere, before use. Oxygen levels were monitored with a 7-ethoxycoumarin was tested by following fluorescence of 7-hy- model 10 gas analyzer (Coy Laboratory Products) and main- droxy coumarin, with excitation at 350 nm and emission at 450 tained Ͻ1 ppm by using a 5% (vol/vol) hydrogen mix in the nm adapted from ref. 26 and TLC. Activity toward ethoxyresoru- presence of a palladium catalyst. fin was determined fluorometrically with excitation at 530 nm Aerobic conditions. The reaction mixture contained 175 nM XplA and emission at 590 nm, adapted from ref. 26. Carbon monoxide ␮ and 150 nM XplB, 50 mM potassium phosphate (pH 6.8), 300 inhibition was carried out aerobically by adding 100 or 500 l per ␮M NADPH, 100 ␮M RDX, 0.72 units Thermoanaerobium ml of carbon monoxide-saturated buffer. brockii alcohol dehydrogenase (Sigma, St. Louis, MO), and 30 ␮l isopropanol in a total volume of 1 ml. All reactions were Plant Transformation Methods. The xplB gene was cloned into the performed at room temperature (20°C), and assays were stopped binary vector pART27 (27) under the control of the CaMV35S by the addition of 10% trichloroacetic acid (240 mg/ml) or, for promoter and ocs terminator, and transformed by Agrobacte- the mass balance experiments, 30-kDa cut-off spin columns rium-mediated floral dipping into wild-type and xplA-expressing (Microcon YM-30; Amicon). The reaction was initiated by the Arabidopsis thaliana, ecotype Columbia-0 as in ref. 9. addition of RDX. Liquid Culture and Soil Leachate Experiments. Liquid culture exper- iments were performed as described (9). Soil leachate studies Analysis of Products. RDX removal was measured by using RP- were carried out on 6-week-old Arabidopsis plants grown under HPLC with a HPLC system (2695 Separations Module and 2996 180 ␮m⅐m2⅐s Ϫ1 light in a 12-h photoperiod. Plants were grown Photodiode Array Detector; Waters) using a Techsphere C18 in pots containing 30 g of uncontaminated soil (Levingtons F2 column (250 ϫ 4.6 mm) under isocratic conditions of 60% water compost), five plants per pot. Each pot was flooded with 50 ml and 40% acetonitrile at a flow rate of 1 ml/min over 10 min. RDX of 180 ␮M RDX, then 7 days later, flushed through with 50 ml elution was monitored at 205 nm, and intergrations were per- of water. The collected soil leachates were analyzed for RDX formed with Empower software. content by using HPLC as described above. Formaldehyde analysis was carried out according to Nash (23). Nitrite analysis followed the method of Scheideler and Ninnemann This work was funded by the Strategic Environmental Research and (24) terminated by removal of proteins with 30-kDa cut-off spin Development Program of the U.S. Department of Defense, the Bio- columns. The concentration of NDAB and MEDINA was deter- technology and Biological Sciences Research Council, and the U.K. mined by using an HPLC system as reported (25). Ministry of Defense.

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Jackson et al. PNAS ͉ October 23, 2007 ͉ vol. 104 ͉ no. 43 ͉ 16827 Downloaded by guest on September 30, 2021