INTRODUCTION

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Bacterial degradation of aromatic compounds under aerobic conditions is often initiated by multicomponent dioxygenase enzyme systems. Since many aromatic compounds are known to be toxic and/or carcinogenic, these bacterial enzymes are important for removing compounds such as benzene, toluene, naphthalene, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and nitroaromatics from the environment. Recently, there has been a great deal of interest in these broad-substrate enzymes for the production of chiral synthons used in the preparation of a wide range of biologically active chemicals and pharmaceuticals, including inositol phosphates, prostaglandins, and antitumor agents (for reviews, see references 5, 7, 24, 35, and 46). The use of these enzymatic routes for the formation of useful products from what were previously considered toxic waste materials is a driving force for the field of "green" chemistry (25).

Two enzyme systems responsible for initiating the degradation of aromatic hydrocarbons are naphthalene dioxygenase (NDO; EC 1.14.12.12) from Pseudomonas sp. strain NCIB 9816-4 and toluene dioxygenase (TDO; EC 1.14.12.11) from Pseudomonas putida F1. The oxygenase components of NDO and TDO have relaxed substrate specificities, and in addition to catalyzing stereospecific cis-dihydroxylation reactions, these enzymes catalyze monooxygenation, desaturation, dealkylation, and sulfoxidation reactions (3, 15, 24, 42, 44, 45). The three enzyme components from each system have been purified and characterized (10, 16, 17, 36, 50, 51), and each set of genes has been cloned, sequenced, and expressed in Escherichia coli (39, 47, 61, 62).

Recent studies have identified two new three-component dioxygenase systems involved in the degradation of nitroaromatic compounds: 2-nitrotoluene dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 (1, 19, 39), and 2,4-dinitrotoluene dioxygenase (DNTDO) from Burkholderia (formerly Pseudomonas) sp. strain DNT (18, 48, 55, 56).

In these three-component enzyme systems, electrons are transferred from NAD(P)H to a flavoprotein reductase, a Rieske [2Fe-2S]-containing ferredoxin, and finally to the terminal oxygenase component, which is also a Rieske iron-sulfur protein. The reduced oxygenase, which was previously designated ISP, catalyzes the stereospecific addition of both atoms of molecular oxygen into the aromatic nucleus of the substrate. As the catalytic portion of the enzyme, the oxygenase determines substrate specificity. The large () subunit contains a Rieske [2Fe-2S] center (29, 53) and a mononuclear iron binding site, which is believed to be the site of oxygen activation (30). Several studies have implicated the  subunit in determining substrate specificity (12, 14, 32, 38, 40, 58). The function of the small () subunit is not known, although reports in the literature have suggested that the  subunit may be involved in substrate specificity in the toluate and biphenyl dioxygenase systems (13, 21, 23).

It is apparent from deduced amino acid sequence comparisons that the  and  subunits of the oxygenase components of the NDO, 2NTDO, and DNTDO systems are closely related to each other and distantly related to those of the TDO system (39, 55, 61). The  subunit of NDO is 84 and 80% identical to the  subunits of 2NTDO and DNTDO, respectively. The  subunit sequences of 2NTDO and DNTDO are 88% identical. The sequence of the  subunit of NDO is 76 and 78% identical to the 2NTDO and DNTDO  subunits, and the  subunits of 2NTDO and DNTDO are 92% identical. In contrast, the amino acid sequence identities of the TDO  and  subunits to the corresponding subunits of the other three enzymes are in the range of 35 and 25%, respectively. Although NDO, 2NTDO, and DNTDO oxygenase components are very similar in amino acid sequence, each enzyme forms a characteristic set of products from a series of substrates (Table 1). All four enzymes convert naphthalene to cis-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol), but the products have different enantiomeric compositions (Table 1). All of the enzymes oxidize 2-nitrotoluene to 2-nitrobenzyl alcohol (45, 55), but 2NTDO forms predominantly 3-methylcatechol from 2-nitrotoluene (19). DNTDO is the only enzyme capable of converting 2,4-dinitrotoluene to 4-methyl-5-nitrocatechol (40, 55). In addition, while NDO and TDO are very effective at converting indole to indigo (11, 62), DNTDO is less effective (55) and 2NTDO carries out the reaction very poorly. These diagnostic substrates allowed us to assess the contributions of the oxygenase  and  subunits to regiospecificity, stereospecificity, and overall substrate range. Hybrid dioxygenases containing various  subunits were generated by using a two-plasmid expression system in E. coli. The genes encoding the  subunits from NDO, 2NTDO, DNTDO, and TDO were individually cloned in standard E. coli expression vectors. The recombinant plasmids were introduced into E. coli strains carrying the reductase, ferredoxin, and  subunit genes from either NDO or DNTDO on a newly constructed compatible expression vector. Following expression in E. coli, the hybrid enzymes were tested for the ability to oxidize the four diagnostic substrates naphthalene, 2-nitrotoluene, 2,4-dinitrotoluene, and indole.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 2. The designations for cloned dioxygenase genes are explained in the footnote to Table 2.

Media and growth conditions. E. coli strains were grown at 37°C in Luria-Bertani medium (9) or Terrific Broth medium (34). Antibiotics were added to the following final concentrations as appropriate: ampicillin, 150 µg/ml; chloramphenicol, 40 µg/ml; tetracycline, 25 µg/ml. To produce induced cells for biotransformation studies, JM109(DE3) strains carrying plasmids of interest were grown at room temperature (approximately 25°C) in minimal medium (MSB) (49) containing 10 mM glucose, 0.1 mM thiamine, ampicillin, and chloramphenicol. Isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 100 µg/ml when the culture turbidity reached 0.6 to 0.8 at 660 nm. After a 2.5-h induction period, the cells were harvested by centrifugation. For plates, MSB was solidified with 1.8% Noble Agar (Difco Laboratories, Detroit, Mich.) and Luria-Bertani medium was solidified with 1.5% Bacto Agar (Difco Laboratories).

Molecular techniques. Plasmid DNA was purified by the method of Lee and Rasheed (34). E. coli strains were transformed by the method of Hanahan (20). Standard molecular biology techniques were used for the preparation and analysis of subclones (2) with E. coli DH5 as the host strain. DNA fragments were purified from gel slices with the GeneClean spin kit as specified by the manufacturer (Bio 101, Vista, Calif.).

Plasmid constructions. A new expression vector, pREP1 (Fig. 1), was constructed as follows. Plasmid pACYC184 was digested with ClaI and HincII to remove the tetracycline resistance gene, and the 0.3-kb ClaI-PvuII fragment containing the multiple-cloning site and T7 promoter from pT7-5 was inserted. Plasmid pDTG162 contains nahAaAbAc under the control of the T7 promoter in pREP1. It was constructed in a two-step procedure by first deleting the SalI fragment carrying the nahAd gene from pDTG141, forming pDTG149, and then inserting the SacI-HindIII fragment of pDTG149, carrying nahAaAbAc, into SacI-HindIII-digested pREP1 to form pDTG162. Plasmid pDTG953 was formed by insertion of the SacI-BamHI fragment of pJS48 carrying dntAaAbAc into the corresponding restriction sites in pREP1. Plasmid pDTG630, a todC2 expression clone, was constructed in a two-step procedure. First pDTG629 was formed by inserting the 0.6-kb EcoRI-BspHI fragment from pDTG613 into EcoRI-NcoI-digested pUCBM21. Then the 0.7-kb EcoRI-PvuII fragment from pDTG629 was inserted into pT7-7 to form pDTG630. Plasmid pDTG850 was constructed by inserting the 4.7-kb SacI-EcoRI fragment of pDTG800 (carrying all of the ntd genes) into SacI-EcoRI-digested pUC13. The ntdAd expression clone, pDTG824, was constructed by inserting the 860-bp MfeI-EcoRI fragment of pDTG850 into EcoRI-digested pUC18. The dntAd expression clone, pDTG951, was formed by digestion of pJS48 with HindIII followed by self-ligation to delete the 4.3-kb HindIII fragment containing dntAa, ORF2, dntAb, and the first one-third of dntAc.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Circular map of expression vector pREP1, a derivative of pACYC184. Cm, chloramphenicol resistance gene, pT7, T7 promoter from vector pT7-5.

Antibody production. Two adult BALB/c AnNHsd mice (Harlan Sprague-Dawley, Indianapolis, Ind.) were immunized with the purified oxygenase component of NDO. Injections (with 115 µg each) were performed subcutaneously on day 1 with the NDO component in Freund's complete adjuvant (Difco Laboratories), subcutaneously on day 21 with the NDO component in Freund's incomplete adjuvant, and intraperitoneally on day 34 with the NDO component in phosphate-buffered saline (12 mM NaK phosphate buffer [pH 7.2], 137 mM NaCl, 2.5 mM KCl). Five days later, the mouse was sacrificed and fusion was carried out by standard procedures (22). Hybridomas were isolated and screened for the production of antibodies specific for the NDO  subunit by enzyme-linked immunosorbent assay (22). One hybridoma that secreted a monoclonal antibody with a strong reaction in Western blot analyses with _NDO was cloned twice by limiting dilution. The isotype was found to be immunoglobulin G2b by using an Isostrip kit (Boehringer Mannheim Corp., Indianapolis, Ind.). At the time of sacrifice, blood was obtained from the mouse by cardiac puncture, and a 1:10,000 dilution of the polyclonal serum showed a strong reaction by enzyme-linked immunosorbent assay with the NDO  and  subunits. Monoclonal antibody 301, which was raised against the  subunit of TDO, was described previously (36).

Indigo formation. JM109(DE3) strains carrying plasmids of interest were grown overnight at 37°C on nitrocellulose filters placed on MSB agar plates containing glucose, thiamine, ampicillin, and chloramphenicol. Dried Whatman no. 1 filter papers that had been soaked in a 10% solution of indole dissolved in acetone were placed in the petri dish covers after colony formation. Production of indigo from indole vapor was observed as the colonies turned blue. No induction was carried out for these studies.

Whole-cell biotransformations. Induced cultures (1 liter) were harvested by centrifugation and resuspended in MSB containing 10 mM glucose (125 ml, final turbidity of 5.0 to 6.0 at 660 nm). Small samples of resuspended cells (four 1-ml samples) were harvested by centrifugation and stored at 20°C for protein determinations and gel electrophoresis. To initiate biotransformation reactions, cell suspensions (40 ml) were added to flasks containing 0.1% (vol/vol) 2-nitrotoluene, 0.1% (wt/vol) naphthalene, or 0.1% (wt/vol) 2,4-dinitrotoluene. Solid substrates were dissolved in acetone, and the acetone was evaporated to leave a thin coating of substrate in the flasks. Cultures were incubated at 30°C with shaking (250 rpm) for up to 24 h. Samples (0.5 ml) were taken periodically, and cells were removed by centrifugation. Culture supernatants were stored at 20°C until analyzed. After 24 h, the cells were removed from the remaining cultures by centrifugation and the culture supernatants were extracted as described below.

Separation and identification of products. High-performance liquid chromatography (HPLC) was used to quantify cis-1,2-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol), 2-nitrobenzyl alcohol, and 4-methyl-5-nitrocatechol in aqueous samples. HPLC analyses were performed with a Waters Corp. (Milford, Mass.) HPLC system equipped with a 600E solvent delivery system, U-6K injector, model 991 photo diode array detector, and Millennium Chromatography Manager software. Metabolites were separated on a Beckman Instruments, Inc. (Fullerton, Calif.) Ultrasphere reverse-phase column (4.6 mm by 25 cm) with a methanol-water mobile phase. Elution was carried out with a linear gradient increasing from 20 to 100% methanol over a 15-min period at a flow rate of 1 ml/min. HPLC analyses for 4-methyl-5-nitrocatechol were performed as described above, except that the water was acidified with trifluoroacetic acid (0.1%, vol/vol). Culture supernatants from 24-h incubations were extracted with sodium hydroxide-washed ethyl acetate and analyzed by thin-layer chromatography (43). All extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) as previously described (44). cis-Naphthalene dihydrodiol was purified for chiral HPLC analysis by preparative-layer chromatography (44). Chiral stationary-phase liquid chromatography was used to resolve the two enantiomers of cis-naphthalene dihydrodiol with a Chirocel OJ column (Chiral Technologies, Exton, Pa.) as described previously (44). Under these conditions, the (+)-(1R,2S) and ()-(1S,2R) enantiomers of cis-naphthalene dihydrodiol eluted with retention times of 30 and 33 min, respectively. Product identifications were based on comparisons to standards.

Chemicals. Naphthalene was obtained from Fisher Scientific Co., Pittsburgh, Pa. Indole, 2-nitrotoluene, 2,4-dinitrotoluene, and 2-nitrobenzyl alcohol were purchased from Aldrich Chemical Co., Milwaukee, Wis. 4-Methyl-5-nitrocatechol was a gift from Jim C. Spain. Synthetic (+/)-cis-naphthalene dihydrodiol and homochiral (+)-cis-naphthalene dihydrodiol were prepared as previously described (26, 41).

Gel electrophoresis and Western blot analyses. Cell pellets (from 1-ml suspensions) were resuspended in 200 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample loading buffer (2) and boiled for 10 min, and the proteins were separated on duplicate sodium dodecyl sulfate-12% polyacrylamide gels (2). One gel was stained with Coomassie blue R-250 to verify that approximately equal amounts of protein were loaded in each lane. The second gel was subjected to Western blotting as described previously (22, 36). Antigens were visualized with alkaline phosphatase conjugated goat anti-mouse immunoglobulin G (Pierce, Rockford, Ill.).

Protein determinations. Cell pellets (from 1-ml suspensions) were resuspended in 0.1 M sodium hydroxide and boiled for 1 h. Protein concentrations were determined by the method of Bradford (4) with bovine serum albumin as the standard.

	RESULTS

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Construction of the two plasmid expression system. A new expression vector, pREP1, was constructed for use in this study (Fig. 1). This plasmid, a derivative of pACYC184, carries a chloramphenicol resistance gene, a T7 promoter, and a multiple-cloning site and is compatible with ColE1 plasmids. In this study, the genes encoding the reductases, ferredoxins, and  subunits from the NDO and DNTDO systems were expressed from pREP1 derivatives (pDTG162 and pDTG953, respectively) in JM109(DE3). Genes encoding  subunits were coexpressed from compatible ColE1 plasmids (Table 2). All genes were inducible either directly or indirectly with IPTG. The gene encoding the NDO  subunit, nahAd, was directly inducible since it is under the control of the lac promoter in pDTG824, a pUC18 derivative. All other genes were under the control of the T7 promoter in plasmids carried in JM109(DE3), a strain that has an IPTG-inducible T7 polymerase gene inserted in the chromosome. Six hybrid enzymes, two wild-type enzymes, and control enzymes containing no  subunit were produced with the two-plasmid expression system (Table 3).

Expression of cloned dioxygenase genes. A monoclonal antibody specific for the  subunit of NDO reacted with the  subunits of NDO, DNTDO, and 2NTDO but not TDO in crude cell extracts (Fig. 2). Similar results were obtained when purified oxygenase components of NDO, 2NTDO, and TDO were analyzed by Western blotting (data not shown). Use of this antibody demonstrated that the  subunits of NDO and DNTDO were produced by all recombinant strains except control JM109(DE3) carrying the two vectors only (Fig. 3A). Polyclonal antiserum raised against NDO reacted with the  subunits of NDO and DNTDO and also with the  subunit of NDO (Fig. 3B, lanes 1 and 8). This polyclonal antiserum was used to verify the production of the  subunit of NDO (lanes 2 and 9). The polyclonal antibody also reacted with a nonspecific E. coli protein present in all crude cell extracts (lanes 2 to 7 and 9 to 14). Monoclonal antibody 301, which was raised against the  subunit of TDO (36), was used to demonstrate the presence of the TDO  subunit in extracts (Fig. 3C). These results show that all oxygenase  and  subunits were present in E. coli extracts with the exception of the  subunits of 2NTDO and DNTDO. Antibodies for the detection of these two  subunits were not available.

View larger version (76K): [in this window] [in a new window]   FIG. 2.   Western blot analysis of crude cell extracts containing various oxygenase components (see Tables 2 and 3 for details of strains). The monoclonal antibody used was specific for NDO (see Materials and Methods). M, molecular mass standards. Lanes: 1, JM109(DE3)(pDTG162)(pDTG124) expressing NDO; 2, DH5(pDTG800) expressing 2NTDO; 3, JM109(pDTG601A) expressing TDO; 4, JM109(DE3)(pDTG953)(pDTG951) expressing DNTDO.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of crude cell extracts of strains producing hybrid dioxygenases (described in Table 3). (A) Monoclonal antibody was specific for NDO. (B) Polyclonal antibody was obtained from a mouse immunized with purified NDO. (C) Monoclonal antibody 301 was specific for TDO (36). M, molecular mass standards. (A and B) Lanes: 1 and 8, purified NDO (3.2 µg each); 2, NDO; 3, NDO-2NTDO; 4, NDO-DNTDO; 5, NDO-TDO; 6, NDO-0; 7, vector control; 9, DNTDO-NDO; 10, DNTDO-2NTDO; 11, DNTDO; 12, DNTDO-TDO; 13, DNTDO-0; 14, vector control. (C) Lanes: 1, purified TDO (5.0 µg); 2, NDO-TDO; 3, NDO-0; 4, DNTDO-TDO; 5, DNTDO-0.

Indigo formation. One rapid qualitative measure of dioxygenase activity is the conversion of indole to indigo. The ability to form indigo by E. coli strains expressing wild-type and hybrid dioxygenases was tested on agar plates. While NDO and TDO have been shown to efficiently convert indole to indigo (11, 62), DNTDO does so less efficiently (55) and 2NTDO only forms trace amounts. The formation of blue colonies by JM109(DE3)(pDTG162)(pDTG824), and JM109(DE3) (pDTG162)(pDTG951), which produce NDO-_2NTDO and NDO-_DNTDO, respectively, was the first indication that these hybrid enzymes were active. None of the other hybrid enzyme-producing strains turned blue. Control strains expressing wild-type NDO and DNTDO formed blue colonies. Strains that lacked a small subunit or contained only vectors remained white.

cis-Naphthalene dihydrodiol formation. Naphthalene was used to test hybrid enzyme activity, since NDO, TDO, 2NTDO, and DNTDO each catalyze the conversion of naphthalene to cis-naphthalene dihydrodiol. The specific activities of wild-type and hybrid enzymes with naphthalene as the substrate are shown in Table 4. Since 2NTDO was produced from a single plasmid, its specific activity cannot be directly compared to the specific activities of the other enzymes. These activities are consistently lower (5- to 10-fold) than are the activities in strains carrying single expression plasmids. One possible reason is that the four genes are no longer coordinately regulated from the same DNA fragment and coupled translation of the  and  subunits cannot occur. A second possibility is that differences in plasmid copy number result in the formation of unequal amounts of  and  gene products. The amounts of cis-naphthalene dihydrodiol formed in 5-h biotransformations are shown in Table 5. NDO-_TDO, DNTDO-_TDO and DNTDO-_NDO did not form detectable amounts of cis-naphthalene dihydrodiol even after prolonged incubation (24 h) as judged by HPLC or GC-MS analyses. cis-Naphthalene dihydrodiol was not formed by control strains that did not contain a  subunit gene.

Stereochemistry of cis-naphthalene dihydrodiol formed by hybrid dioxygenases. The stereochemistry of the cis-naphthalene dihydrodiol formed by hybrid dioxygenases was determined by chiral stationary-phase HPLC. NDO forms enantiomerically pure (+)-(1R,2S)-cis-naphthalene dihydrodiol from naphthalene (27, 28). DNTDO and 2NTDO form characteristic ratios of the (+) and () enantiomers of cis-naphthalene dihydrodiol (40, 55). The results obtained with hybrid enzymes indicated that the  subunit does not play a role in determining the enantiomeric purity of the cis-naphthalene dihydrodiol formed (Table 4).

Products formed from 2-nitrotoluene and 2,4-dinitrotoluene. While all of the wild-type oxygenases in this study are capable of forming 2-nitrobenzyl alcohol from 2-nitrotoluene (33, 45, 55), only 2NTDO forms 3-methylcatechol from this substrate (19). Two NDO hybrid enzymes (NDO-_2NTDO and NDO-_DNTDO) formed 2-nitrobenzyl alcohol from 2-nitrotoluene (Table 5) but formed no 3-methylcatechol as judged by HPLC and GC-MS analyses. No oxidation products were formed from 2-nitrotoluene by the other hybrid enzymes or by the control strains carrying no  subunit gene (Table 5). From these results, the  subunit does not appear to determine the position of attack (regiospecificity) on 2-nitrotoluene, since neither NDO-_2NTDO nor DNTDO-_2NTDO was capable of converting 2-nitrotoluene to 3-methylcatechol.

	DISCUSSION

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The oxygenase subunits of three of the four dioxygenases used in this study are very similar in amino acid sequence, yet these enzyme systems have significant and easily measurable differences in substrate specificity. These characteristics have made the study of hybrid enzymes useful in assessing the role of the subunit in substrate specificity. Of the six hybrids constructed, three were active. The hybrid enzymes NDO-_TDO and DNTDO-_TDO were not active with any of the substrates tested. This was not surprising since _TDO is only 22 and 24% identical in amino acid sequence to _NDO and _DNTDO, respectively. It is possible that the structure of _TDO differs from those of _NDO and _DNTDO and that for this reason it cannot interact effectively with the subunits of either NDO or DNTDO to form an active oxygenase. It is interesting that DNTDO-_NDO was not functional but DNTDO-_2NTDO was quite active (Table 5), especially since _NDO is slightly more similar in sequence to _DNTDO than to _2NTDO. Western blot analyses showed that the  and  subunits of the terminal oxygenase were present in cell extracts of strains expressing all three inactive hybrids. Therefore, the absence of enzymatic activity was not due to problems in protein production. We were unable to test for production of reductase and ferredoxin in the inactive strains, but presumably these proteins were produced, since control wild-type and active hybrid enzymes were generated with the same plasmid constructs. With the two-plasmid expression system used, the only difference between the two sets of strains for production of hybrid NDO and DNTDO enzymes was in the plasmid carrying the  subunit gene.

The  subunits of various dioxygenases are important in controlling substrate specificity. Although the oxygenase components of biphenyl dioxygenase from strains KF707 and LB400 are very similar (20 and 1 amino acid differences in the  and  subunits, respectively), the two enzymes have very different polychlorinated-biphenyl congener specificities (14). Detailed studies have indicated that the  subunits are responsible for these differences in substrate specificity (12, 32, 38).

Reports in the literature have suggested that the  subunit may play a role in determining substrate specificity (13, 21, 23). In one study, a mutation that resulted in a broad substrate toluate dioxygenase mapped to the  subunit gene (21). However, a detailed characterization of this mutant has not been reported. In another series of studies, hybrid biphenyl dioxygenase enzymes in which the biphenyl dioxygenase  subunit was replaced with the toluene dioxygenase  subunit were constructed. The resulting enzyme was reported to have an extended substrate range which included the ability to convert benzene and toluene to the corresponding cis-dihydrodiols (13, 23). It appeared that these hybrid enzymes formed more stable quaternary complexes than did wild-type biphenyl dioxygenase (23). In a similar study, a more sensitive assay indicated that wild-type biphenyl dioxygenase did have the ability to oxidize benzene, and the investigators concluded that the subunit alone was critical for substrate specificity (58). Taken together, these results suggest that the hybrid enzymes might have been slightly more active due to improved enzyme stability and that the  subunit was not actually conferring new catalytic activities. Recent work in our laboratory indicated that the substrate specificity of 2NTDO was not affected when its  subunit was replaced by the  subunit from DNTDO (40). The present study indicates that the  subunit alone determines substrate specificity and the  subunit does not contribute significantly to any aspect of specificity, including substrate range, regiospecificity, and stereospecificity.

These results leave us still searching for a function for the  subunit. In a recent review article, the authors suggested that the  subunit of the benzene dioxygenase terminal component might be involved in ferredoxin docking and electron transfer, based on results of unpublished cross-linking studies (6). However, work with the closely related toluene dioxygenase system showed that the Rieske center in the purified TDO  subunit could be enzymatically reduced in the presence of NADH and catalytic amounts of reductase_TOL and ferredoxin_TOL, indicating that the  subunit is not required for electron transfer from ferredoxin_TOL (29). However, the  subunit was required for product formation, and active TDO could be readily reconstituted from separately purified  and  subunits.

The function of the small subunit may be primarily structural. The crystal structure of NDO (31) indicates that the  subunit does not appear to be located at the predicted active site and that only a small portion of the  subunit interacts with the Rieske center domain of the  subunit. From the crystal structure and analytical ultracentrifugation analyses, the native conformation of NDO was found to be an ₃₃ hexamer (31). The three  subunits may function to hold the three  subunits in place. This conformation is apparently essential, since the active site appears to be located at the junction between adjacent  subunits (31).