INTRODUCTION

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Environmental contamination by nitroaromatic compounds is largely due to their extensive use in the production of dyes, pesticides, herbicides, and explosives. This problem is further compounded by the resistance of nitroaromatic compounds to biodegradation. The recalcitrant nature of nitroaromatic compounds is due in large part to the strong electron-withdrawing property of nitro groups, which causes the aromatic nucleus of nitroaromatic compounds to be electron deficient and thereby resistant to electrophilic attack by oxygenases (22). The ability to remove nitro groups would therefore greatly enhance an organism's ability to degrade nitroaromatic compounds.

Pseudomonas sp. strain JS42 was isolated by virtue of its ability to use 2-nitrotoluene (2NT) as the sole source of carbon and nitrogen (8). The initial reaction in the biodegradation of 2NT by JS42 requires molecular oxygen for the conversion of 2NT to 3-methylcatechol and is accompanied by the release of the nitro group as nitrite. This reaction is catalyzed by the three-component dioxygenase system 2-nitrotoluene (2NT) 2,3-dioxygenase (2NTDO), which adds both atoms of molecular oxygen to the aromatic nucleus of 2NT (1). The initial step in the biodegradation of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT) is the conversion of 2,4-DNT to 4-methyl-5-nitrocatechol and nitrite (25). This reaction is also catalyzed by a three-component dioxygenase system, 2,4-dinitrotoluene (DNT) dioxygenase (2,4-DNTDO), and is analogous to the reaction catalyzed by 2NTDO. Both dioxygenase systems consist of an iron-sulfur flavoprotein reductase and an iron-sulfur ferredoxin which transfer electrons to a terminal oxygenase (10). The terminal oxygenase components of these enzymes are iron-sulfur proteins (ISPs) which consist of two dissimilar subunits (ISP and ISP). The genes encoding 2,4-DNTDO and 2NTDO have recently been cloned and sequenced (19, 28, 29).

We now report studies on the specificity of 2NTDO and hybrid dioxygenases. These results are compared to those of 2,4-DNTDO as well as naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4, since all of these enzymes catalyze the conversion of naphthalene to cis-1,2-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol). Results indicate that only the C-terminal region of the ISP subunit of 2NTDO is responsible for the specificity differences observed between 2NTDO and 2,4-DNTDO.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Recombinant Escherichia coli strains were maintained on agar plates containing minimal salts medium (MSB) (26), 0.8% (wt/vol) agar, 10 mM glucose, 1 mM thiamine, and ampicillin (200 µg/ml). Strains used for biotransformations were DH5(pUC18), DH5(pDTG800), DH5(pDTG832), DH5(pDTG833), DH5(pDTG834), JM109(DE3) (pDTG141), and JM109(DE3)(pJS48). Cells were grown aerobically at 37°C in 2-liter Fernbach flasks containing 750 ml of MSB supplemented with 10 mM glucose, 1 mM thiamine, and ampicillin (200 µg/ml). During log-phase growth (A₆₆₀  0.8), the temperature was lowered to 30°C and the cloned dioxygenase genes were induced by addition of isopropyl--D-thiogalactopyranoside (IPTG) to a final concentration of 100 µM. The cultures were then incubated for an additional 2 to 3 h, harvested by centrifugation, and resuspended in MSB medium, pH 7.3 (supplemented with 10 mM glucose) to an A₆₆₀ of 2.0 to 2.5. Dioxygenase activity in cultures of E. coli strains carrying pDTG800, pDTG832, pDTG833, and pDTG834 decreased after the addition of IPTG (possibly due to the formation of inclusion bodies). Therefore, these cultures were grown as described above but without the addition of IPTG.

Biotransformations of aromatic substrates. Cell suspensions for biotransformation experiments were prepared as described above and added to flasks containing 10 mM glucose and 0.1% (wt/vol) of the specified substrate. The flasks were incubated with shaking at 30°C for 7 h, after which time cells were removed by centrifugation. Whole-cell protein was determined by resuspending cell pellets in 100 mM NaOH, boiling for 10 min, and determining the protein concentration as previously described (3) with bovine serum albumin as the standard. Biotransformation products were extracted from the clarified supernatant with ethyl acetate as previously described (21).

Analysis of biotransformation products. Biotransformation products were analyzed by gas chromatography-mass spectrometry as previously described (21) and high-performance liquid chromatography (HPLC). HPLC analyses were performed with a Waters Associates HPLC system (600E solvent delivery system, U-6K injector, model 910 photo diode array multiwavelength detector, and Millennium Chromatography Manager software). HPLC separations were carried out on a Beckman Ultrasphere reverse-phase column (4.6 by 25 cm) with the following conditions. HPLC method 1 utilized a 0.1% aqueous trifluoroacetic acid (TFA)-acetonitrile mobile phase. The concentration of acetonitrile was increased using a linear gradient from 0 to 75% over a 25-min time period and held at 75% for an additional 10 min. Method 2 utilized a TFA-methanol mobile phase. The initial methanol concentration was held at 0% for 10 min, after which it was increased using a linear gradient to 40% over 110 min. The methanol concentration was then raised to 100% for an additional 10 min. Method 3 utilized a TFA-methanol mobile phase in which the methanol concentration was increased from 0 to 100% over a 30-min time period and held at 100% for an additional 5 min. Method 4 utilized a water-methanol mobile phase. The methanol concentration was increased using a linear gradient from 20 to 100% over a 15-min period and maintained at 100% for an additional 5 min. Flow rates for all methods were 1 ml/min. cis-Naphthalene dihydrodiol was purified by preparative thin-layer chromatography using 0.2-mm-thick Silica Gel 60F₂₅₄ plates (EM Separations Technology, Gibbstown, N.J.) as previously described (21). The enantiomeric composition of cis-naphthalene dihydrodiol was determined by chiral stationary-phase HPLC using a Chiracel OJ column (Chiral Technologies, Inc., Exton, Pa.) as described previously (21). Under these conditions the (+)-(1R,2S) and the ()-(1S,2R) enantiomers of cis-naphthalene dihydrodiol eluted at retention times of approximately 30 and 33 min, respectively.

DNA purification and manipulations. Plasmid DNA was isolated by the method of Lee and Rasheed (16). DNA manipulations were performed by standard procedures (2). DNA fragments were purified from agarose gel slices using the Gene Clean II system as specified by the manufacturer's directions (Bio 101, Inc., Vista, Calif.). E. coli DH5 was made competent and transformed with recombinant plasmid DNA by the method of Hanahan (9). Nucleotide and amino acid sequence analyses were performed by using the Wisconsin Sequence Analysis package (Genetics Computer Group, Madison, Wis.).

Chemicals. The following chemicals were obtained from the sources indicated: nitrobenzene, Mallinckrodt, Paris, Ky.; naphthalene, Fisher Scientific Co., Pittsburgh, Pa.; 3- and 4-methylcatechol, Pfaltz and Bauer, Inc., Waterbury, Conn.; and 2NT, 3NT, 4NT, 2,4-DNT, 2-, 3-, and 4-nitrobenzyl alcohol, and catechol, Aldrich Chemical Co., Milwaukee, Wis. 4-Methyl-5-nitrocatechol was supplied by Jim C. Spain (Tyndall Air Force Base, Fla.). Synthetic (±)-cis-1,2-dihydroxy-1,2-dihydronaphthalene and homochiral (+)-(1R,2S)-cis-1,2-dihydroxy-1,2-dihydronaphthalene were prepared as previously described (13, 20).

	RESULTS

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Substrate specificities of 2NTDO, 2,4-DNTDO, and NDO. A comparison of the substrates oxidized by recombinant E. coli strains expressing 2NTDO, 2,4-DNTDO, and NDO is shown in Table 2. 2NTDO catalyzed the dihydroxylation of the aromatic nucleus of 2NT, 3NT, 4NT, and nitrobenzene. Small amounts of 2-, 3-, and 4-nitrobenzyl alcohol were also formed from 2NT, 3NT, and 4NT, respectively. 2,4-DNTDO formed the benzylic alcohol derivatives of all nitrotoluene isomers. In addition, a small amount of the dioxygenation product of 4NT was formed. Similarly, NDO only catalyzed the benzylic hydroxylation of 2NT, 3NT, and 4NT. Neither 2,4-DNTDO nor NDO formed oxidation products from nitrobenzene. All three dioxygenases catalyzed the dihydroxylation of naphthalene to form cis-naphthalene dihydrodiol. Of these three dioxygenases, only 2,4-DNTDO catalyzed the dihydroxylation of the aromatic nucleus of 2,4-DNT. None of these products were detected in biotransformations with E. coli carrying the vector only.

Comparisons of nucleotide and deduced amino acid sequences of 2NTDO, 2,4-DNTDO, and NDO. A high level of nucleotide and deduced amino acid sequence identity exists between the individual components of the three dioxygenases. Of particular interest is the high level of deduced amino acid sequence identity (88%) and nucleotide sequence identity (95%) between ISP from 2NTDO and 2,4-DNTDO. An alignment of the deduced amino acid sequences for the ISP subunits from 2NTDO, 2,4-DNTDO, and NDO is shown in Fig. 1. The results described above indicated that 2,4-DNTDO and NDO have similar substrate specificities which differ significantly from that of 2NTDO. This observation led us to locate 12 positions in the ISP_2NT deduced amino acid sequence that differ from the deduced amino acid sequences of both ISP_DNT and ISP_NAP. Eleven of these amino acids are clustered in the C-terminal region of ISP_2NT (Fig. 1). This region is downstream of conserved histidine and cysteine residues involved in binding the Rieske [2Fe-2S] center (4, 18, 23) and the amino acids thought to be involved in coordinating mononuclear iron at the active site of the enzyme (14).

View larger version (69K): [in this window] [in a new window]   FIG. 1.   Alignment of deduced amino acid sequences from ISP2NT (19), ISPDNT (28), and ISPNAP (19, 24). Differences in the ISP amino acid sequences which may be responsible for the observed differences in substrate specificities between 2NTDO and 2,4-DNTDO are denoted by circled numbers. Conserved histidine and cysteine residues coordinating the [2Fe-2S] Rieske cluster are denoted by asterisks. Conserved amino acids thought to coordinate mononuclear iron are denoted by inverted triangles.

Construction of hybrid dioxygenases. Conserved KpnI and MfeI restriction sites were located in the nucleotide sequences of the genes ntdAc and dntAc, which encode ISP_2NT and ISP_DNT, respectively (Fig. 2). These restriction sites flank the region of DNA encoding the portion of the C-terminal region of ISP_2NT where the 11 amino acids of interest are clustered. The KpnI/MfeI DNA fragment was isolated from pJS48, which carries the genes encoding 2,4-DNTDO (28), and was used to replace the analogous DNA fragment in pDTG800. The latter plasmid carries the genes encoding 2NTDO (19). The new plasmid was designated pDTG832. To determine if ISP and ISP interactions were of importance in 2NTDO substrate specificity, the 1.6-kb MfeI/XbaI DNA fragments (containing the gene encoding ISP_2NT) from both pDTG800 and pDTG832 were replaced by the analogous MfeI/XbaI DNA fragment from pJS48 (containing the gene encoding ISP_DNT). The resulting plasmids were designated pDTG834 and pDTG833, respectively. Gene organization and partial restriction maps of pDTG800, pDTG832, pDTG833, pDTG834, and pJS48 are shown in Fig. 2. E. coli strains transformed with these plasmids were used in the biotransformation studies described below. The hybrid dioxygenase enzymes produced by DH5 carrying pDTG832, pDTG833, and pDTG834 were designated HD832, HD833, and HD834, respectively.

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Gene organization and partial restriction maps of pJS48 (genes encoding 2,4-DNTDO [DNTDO]), pDTG800 (genes encoding 2NTDO), pDTG832 (genes encoding HD832), pDTG833 (genes encoding HD833), and pDTG834 (genes encoding HD834). Shaded areas indicate the relevant DNA from the genes expressing 2,4-DNTDO.

Biotransformation of naphthalene by hybrid dioxygenases. The three dioxygenases 2NTDO, 2,4-DNTDO, and NDO oxidize naphthalene to cis-naphthalene dihydrodiol. Consequently the biotransformation of naphthalene was used as a diagnostic tool to determine if active enzymes were expressed by the hybrid gene clusters. Biotransformation experiments demonstrated that all three hybrid dioxygenases oxidized naphthalene to cis-naphthalene dihydrodiol. Results from time course studies show that 2NTDO and HD833 produce cis-naphthalene dihydrodiol at a much higher rate (0.48 and 1.22 µg/min/mg of total protein, respectively) than either HD832 or HD834 (0.07 µg/min/mg of total protein each).

Substrate specificity of hybrid dioxygenases. The ability of the hybrid dioxygenases to oxidize the substrates listed in Table 2 was determined. The results show that HD832 did not catalyze the dihydroxylation of 2NT, 3NT, 4NT, or nitrobenzene. The enzyme did, however, catalyze the oxidation of the methyl substituents of 2NT, 3NT, and 4NT. In addition, HD832 gained the ability to catalyze the dihydroxylation of 2,4-DNT to 4-methyl-5-nitrocatechol. Thus, replacing the C-terminal region of ISP_2NT with the corresponding region from ISP_DNT resulted in a change in the substrate specificity and regiospecificity of 2NTDO. Replacement of the ISP subunit in HD832 or 2NTDO with the corresponding subunit from 2,4-DNTDO (HD833 and HD834, respectively) resulted in no change in substrate specificity or regiospecificity (Table 2). These results show that the ISP subunit does not play a role in determining substrate specificity or regiospecificity.

Enantiomeric purity of cis-naphthalene dihydrodiols formed by 2NTDO and hybrid dioxygenases. The enantiomeric compositions of the cis-naphthalene dihydrodiols formed by 2NTDO, 2,4-DNTDO, NDO, and the hybrid dioxygenases were determined. The results show that the enantiomeric composition of the cis-naphthalene dihydrodiol formed by 2NTDO is 70% (+)-(1R,2S). In contrast, the enantiomeric compositions of the cis-naphthalene dihydrodiols formed by 2,4-DNTDO and NDO were 96 and >99% (+)-(1R,2S) respectively. The enantiomeric composition of the cis-naphthalene dihydrodiol formed by the hybrid dioxygenase HD832 was 98% (+)-(1R,2S). These results clearly indicate that the C-terminal region of ISP_2NT plays a major role in determining the enantiomeric compositions of the products formed by 2NTDO. The enantiomeric compositions of the cis-naphthalene dihydrodiols formed by HD833 and HD834 were also determined and found to be 96 and 70% (+)-(1R,2S), respectively, indicating that the ISP subunit of the terminal oxygenase of 2NTDO does not influence the enantiomeric compositions of the products formed by 2NTDO.

	DISCUSSION

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Recent studies have demonstrated that 2,4-DNTDO and NDO exhibit a high level of deduced amino acid sequence identity and oxidize many of the same compounds (28). Based on the high level of nucleic acid and amino acid sequence identity between 2NTDO and 2,4-DNTDO, one would expect 2NTDO and 2,4-DNTDO to have similar substrate oxidation profiles. Yet this is not the case. We have shown that 2NTDO is capable of catalyzing the dihydroxylation and/or monohydroxylation of several aromatic compounds and that the substrate oxidation profile of 2NTDO is different from that of 2,4-DNTDO. Furthermore, we have shown that these observed differences in enzyme specificity can be attributed to differences in the amino acid sequence of the C-terminal regions of ISP_2NT and ISP_DNT. This is the first report demonstrating that the enantiomeric composition of the products formed by a bacterial multicomponent dioxygenase is determined by the C-terminal region of the ISP subunit of the oxygenase component.

The results reported here are in agreement with earlier studies which have shown that replacing the ISP subunit of biphenyl dioxygenase (BPDO) from Pseudomonas pseudoalcaligenes KF707 with the ISP subunit from toluene dioxygenase (TDO) results in a hybrid biphenyl dioxygenase with altered substrate specificity more similar to that of TDO than BPDO (6, 7, 12). The substrate specificity of BPDO has also been altered by replacing the ISP subunit of BPDO with the ISP subunit of benzene dioxygenase from Pseudomonas putida ML2 (31). The resulting hybrid dioxygenase gained the ability to catalyze the transformation of indole to indigo, a reaction not catalyzed by BPDO. Erickson and Mondello (5) have shown that changing four amino acids in the C-terminal region of the ISP subunit of the Pseudomonas sp. strain LB400 biphenyl dioxygenase resulted in a dioxygenase with an expanded polychlorinated biphenyl congener specificity. More recent studies have shown that changes in substrate specificity of BPDO from P. pseudoalcaligenes KF707 and Pseudomonas sp. strain LB400 can be attributed to a single amino acid change in the C-terminal region of ISP (15, 17). These studies clearly demonstrate the importance of the ISP subunit in determining the specificity of these dioxygenases.

The role of the ISP subunit is less clear. Earlier studies have indicated that the ISP subunit may play a role in determining the substrate specificity of toluate dioxygenase (11). Similar results have also been demonstrated for BPDO and TDO (12). Results presented here clearly indicate that ISP does not play a role in determining substrate specificity for 2NTDO or 2,4-DNTDO. However, higher yields of oxidation products were observed in biotransformations with 2NTDO and HD833 than with HD832 and HD834 (Table 2), suggesting that the ISP subunit may interact with the C-terminal region of the ISP subunit. Further work will be necessary to confirm this conclusion.

Experiments are under way to identify specific amino acids which are responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. These and future studies may contribute insights to factors which influence and/or control the enzyme specificity of bacterial multicomponent dioxygenases.