INTRODUCTION

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The naphthalene dioxygenase (NDO) system (EC 1.14.12.12) catalyzes the first step in the degradation of naphthalene in Pseudomonas sp. NCIB 9816-4. In this reaction, both atoms of O₂ are added to the aromatic ring to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-dihydrodiol) (28, 29). NDO consists of three components. An iron-sulfur flavoprotein reductase and a Rieske iron-sulfur ferredoxin transfer electrons from NAD(P)H to the catalytic oxygenase component (15, 16, 21, 22). The oxygenase consists of large () and small () subunits that form an ₃₃ native structure (31). Each  subunit contains a Rieske [2Fe-2S] center and mononuclear nonheme iron (15, 31). Electrons are transferred from the Rieske center in one  subunit to the mononuclear iron in an adjacent  subunit (31, 39), and this is the site of oxygen activation and catalysis.

NDO catalyzes the oxidation of a wide variety of aromatic compounds, and many of the products are enantiomerically pure chiral compounds (9, 24, 45). The use of dioxygenases to initiate biocatalytic routes for the production of pharmaceuticals and natural products has received significant attention of late (9, 12, 25, 42), and the possibility of generating new synthons with opposite stereochemistry is an attractive alternative to asymmetric chemical synthesis (7).

From the crystal structure of NDO, several amino acids were identified near the active site (31). Site-directed mutagenesis at nine positions near the mononuclear iron identified Phe-352 as an amino acid that plays an important role in determining the regioselectivity of biphenyl and phenanthrene oxidation and the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene (38). Valine and leucine substitutions at Phe-352 resulted in the largest specificity changes with several substrates. In this study, we generated and characterized six new enzymes with amino acid substitutions at Phe-352. Results show that Phe-352 controls enantioselectivity with naphthalene, biphenyl, phenanthrene, and anthracene as substrates. A previously undescribed compound, the ()-enantiomer of biphenyl cis-3,4-dihydrodiol was identified and characterized.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains DH5 and JM109(DE3) were used for subcloning and gene expression studies, respectively. Competent E. coli strains ES1301 and JM109 were purchased from Promega Corp., Madison, Wis., for use in generating site-directed mutants as described below.

Media and growth conditions. Sphingomonas yanoikuyae (previously Beijerinckia sp.) B8/36 was grown at 30°C in minimal salts medium (MSB) (51) containing 40 mM pyruvate and 0.1% yeast extract. Cultures were induced for 8 h with m-xylene provided in the vapor phase. Pseudomonas putida F1 was grown in MSB with 40 mM pyruvate and toluene provided in the vapor phase. Except where indicated below, E. coli strains were grown at 37°C in Luria-Bertani medium (14) or Terrific Broth medium (34). For growth of E. coli strains harboring plasmids, antibiotics were added to the following final concentrations as appropriate: ampicillin, 150 µg/ml; tetracycline, 12.5 µg/ml; or kanamycin, 50 µg/ml. For biotransformation studies, E. coli JM109(DE3) strains carrying plasmids of interest were grown at 30°C in MSB containing 10 mM glucose, 0.1 mM thiamine, and the appropriate antibiotics. Isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 100 µM when culture turbidity reached 0.6 to 0.8 at 660 nm. After a 2-h induction, biotransformations were initiated as described below. To produce solid media, MSB was solidified with 1.8% Noble agar (Difco Laboratories), and LB was solidified with 1.5% Bactoagar (Difco Laboratories).

Whole-cell biotransformations. After induction, E. coli cultures were supplemented with 20 mM glucose and 80 mM phosphate buffer (pH 7.2). Solid substrates (naphthalene, biphenyl, or phenanthrene) were added to a final concentration of 0.025% (wt/vol). Tween 20 (0.005% [vol/vol]; Aldrich Chemical Co., Milwaukee, Wis.) was added to biotransformations with anthracene (0.0125% [wt/vol]) to increase substrate availability. Cultures were incubated at 30°C with shaking (250 rpm) for 15 to 18 h. Control biotransformations carried out under identical conditions with JM109(DE3)(pT7-5) did not result in product formation with any of the substrates tested. Large-scale (5.5-liter) biotransformations with biphenyl or phenanthrene were carried out in a 10-liter Biostat B fermentor. Induced cultures were incubated at 27°C for 14 to 17 h with 0.025% (wt/vol) substrate, high agitation (700 rpm), automated pH control (pH 7.3), and a slow glucose feed. S. yanoikuyae B8/36 biotransformations were carried out at 30°C with induced cultures (800 ml) supplemented with 40 mM pyruvate-0.025% (wt/vol) substrate.

Rates of product formation. Cultures (50 ml in 500-ml flasks) were grown and induced, and biotransformations with naphthalene or biphenyl were initiated as described above. Samples (1 ml each) were taken at 30-min intervals over a period of 5 h. Cells were removed by centrifugation, and pellets were stored at 20°C for protein determinations. Naphthalene cis-dihydrodiol formation was monitored at 262 nm ( = 8114 M¹ cm¹ [28]). Biphenyl cis-2,3-dihydrodiol formation was monitored at 303 nm ( = 13,600 M¹ cm¹ [19]). Biphenyl cis-3,4-dihydrodiol formation was monitored at 276 nm ( = 4340 M¹ cm¹ [38]) using a correction for the absorbance of biphenyl cis-2,3-dihydrodiol at this wavelength. The extinction coefficient of biphenyl cis-2,3-dihydrodiol at 276 nm (the _max of biphenyl cis-3,4-dihydrodiol) was determined to be 7950 M¹ cm¹ using purified biphenyl cis-2,3-dihydrodiol produced by S. yanoikuyae B8/36 (19). The concentration of biphenyl cis-3,4-dihydrodiol was calculated using the ratios of products formed by each mutant enzyme (see results) and subtracting the contribution of biphenyl cis-2,3-dihydrodiol. Absorbance readings obtained from control naphthalene or biphenyl biotransformations with JM109(DE3)(pT7-5) were subtracted at each time point to eliminate the contributions of the substrates as they dissolved. Protein concentrations were determined by the method of Bradford (10) after boiling cell pellets for 1 h in 0.1 N NaOH. Bovine serum albumin was used as the standard. Reported rates are the averages of three independent experiments.

Indigo formation. E. coli JM109(DE3) strains carrying plasmids of interest were grown overnight at 37°C on nitrocellulose filters placed on MSB agar plates containing glucose, thiamine, and ampicillin. Whatman no. 1 filter papers were soaked in a 10% solution of indole dissolved in acetone, dried, and placed in the covers of inverted petri dishes after colony formation. Production of indigo from indole vapor was observed as colonies turned blue. Cultures were not induced.

Molecular techniques. Plasmid DNA was purified as previously described (34) or with the Qiagen Midi Kit (Qiagen, Inc., Santa Clarita, Calif.). DNA to be used for nucleotide sequencing was further purified using a Centricon-100 filter unit (Amicon, Inc., Beverly, Mass.). Restriction digests were performed as suggested by the enzyme suppliers (New England Biolabs, Inc., Beverly, Mass.; Promega Corp.). DNA fragments were purified from gel slices using the GeneClean Spin Kit according to the manufacturer's instructions (BIO101, Vista, Calif.). Ligation reactions, E. coli transformations, and agarose gel electrophoresis were performed by standard methods (49).

Site-directed mutagenesis. Mutagenesis of nahAc was carried out with the Altered Sites II in vitro mutagenesis system according to the manufacturer's instructions (Promega Corp.). Plasmid pMASTER-1 (39) carries the 3' end of the nahAc gene and the complete nahAd gene (encoding the  and  subunits of NDO, respectively) and was used as the template for mutagenesis. Each mutagenic oligonucleotide (Table 2) was designed with a silent mutation that altered the restriction pattern of the plasmid (eliminating an AclI site) to facilitate mutant screening. Phosphorylated oligonucleotides used for mutagenesis were synthesized by Genosys Biotechnologies Inc., Midland, Tex. The nucleotide sequences of both strands of each insertion in pMASTER-1 were determined for each mutant. Fluorescent, automated DNA sequencing was carried out at the University of Iowa DNA Facility using an Applied Biosystems 373A automated DNA sequencer. The 1.5-kb KpnI-XbaI fragments carrying each mutation were individually cloned into KpnI-XbaI-digested pDTG141, and the resulting plasmids were introduced into E. coli strain JM109(DE3) for expression studies. After this subcloning step, the presence of each mutation was verified by restriction and sequence analyses.

Separation and identification of products. Culture supernatants from whole-cell biotransformation experiments were extracted with sodium hydroxide-washed ethyl acetate and analyzed by thin-layer chromatography (TLC) (47). Phenyl boronic acid derivatives (23) were prepared and analyzed by gas chromatography-mass spectrometry (GC-MS) as previously described (44). Naphthalene cis-dihydrodiol and anthracene cis-dihydrodiol were purified by preparative-layer chromatography (PLC) with chloroform-acetone (8:2) (44). Regioisomers of biphenyl cis-dihydrodiol were separated by PLC or radial-dispersion chromatography using a Chromatotron (Harrison Research, Palo Alto, Calif.) as previously described (38).

Chemicals. Naphthalene was obtained from Fisher Scientific Co., Pittsburgh, Pa. Indole, biphenyl, phenanthrene, and anthracene were purchased from Aldrich Chemical Co. 3,4-Dihydroxybiphenyl was obtained from Ultra Scientific, North Kingstown, R.I. Synthetic (±)-naphthalene cis-dihydrodiol and homochiral (+)-naphthalene cis-dihydrodiol were prepared as previously described (27-29). The (+)-enantiomer of biphenyl cis-2,3-dihydrodiol was produced by S. yanoikuyae B8/36 (19) and the ()-enantiomer by Pseudomonas stutzeri C250 (43). Synthetic phenanthrene cis-9,10-dihydrodiol having the reported (50) characteristics (mp 170 to 171°C) was prepared using the method previously described for K-region cis-dihydrodiols of chrysene, benzo[c]phenanthrene and 7,12-dimethylbenz[a]anthracene (5). (+)-Anthracene cis-1,2-dihydrodiol was prepared as previously described using S. yanoikuyae B8/36 (30).

Synthesis of (+)-biphenyl cis-3,4-dihydrodiol. (+)-Biphenyl cis-3,4-dihydrodiol [(+)-cis(1S,2R)-1,2-dihydroxy-4-phenylcyclohexa-3,5-diene] was synthesized in a five-step procedure as described below and as outlined in Fig. 1. 1-Bromo-2-iodobenzene (2.0 g, 7.07 mmol [compound 1]) was oxidized by P. putida UV4 using the standard procedure (7, 8), to yield the major cis-diol, (+)-cis-(1S,2R)-1,2-dihydroxy-4-bromo-3-iodocyclohexa-3,5-diene (compound 2) (0.8 g, 36%), mp, 101 to 103°C (EtOAc-hexane), []_D + 75 (c 2.96, MeOH), _H (500 MHz, CDCl₃), 4.38 (d, 1H, J_2,1 5.9, H-2), 4.48 to 4.50 (m, 1H, H-1), 5.98 (dd, 1H, J_5,6 9.7, J_5,1 3.5, H-5), 6.07 (dd, 1H, J_6,1 1.4, J_6,5 9.7, H-6); found C, 22.6; H, 1.7; C₆H₆IBrO₂ requires C, 22.7; H, 1.9. 

View larger version (5K): [in this window] [in a new window]   FIG. 1.   Strategy for the synthesis of (+)-biphenyl cis-3,4-dihydrodiol. Compound 1, 1-bromo-2-iodobenzene; compound 2, (+)-cis-(1S,2R)-1,2-dihydroxy-4-bromo-3-iodocyclohexa-3,5-diene; compound 3, (+)-cis-(1S,2R)-1,2-dihydroxy-4-bromocyclohexa-3,5-diene; compound 4, ()-cis-(1S,2R)-1,2-di-tert-butyldimethylsilyloxy-4-bromocyclohexa-3,5-diene; compound 5, (+)-cis-(1S,2R)-1,2-di-tert-butyldimethylsilyloxy-4-phenylcyclohexa-3,5-diene; compound 6, (+)-cis-(1S,2R)-1,2-dihydroxy-4-phenylcyclohexa-3,5-diene [(+)-biphenyl cis-(3R,4S)-dihydrodiol].

Gel electrophoresis and Western blot analyses. Cell pellets (from 1-ml suspensions) were resuspended in 200 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (2) and boiled for 10 min, and proteins (approximately 20 µg of total protein per lane) were separated by SDS-12% PAGE (2). The gel was subjected to Western blotting as described previously using a monoclonal antibody specific for the  subunit of NDO (22, 36). Antigens were visualized using alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (Pierce, Rockford, Ill.).

Partial purification of toluene cis-dihydrodiol dehydrogenase. Toluene-grown cells of P. putida F1 were harvested by centrifugation (10 min, 8,000 × g, 4°C) and resuspended in Tris buffer (50 mM Tris-HCl [pH 7.2]-20 µg of DNase I per ml-1.0 mM phenylmethylsulfonyl fluoride). Cell extracts were prepared by passage of cell suspensions through a chilled French pressure cell (20,000 lb/in²), followed by ultracentrifugation (40,000 × g, 60 min, 4°C). Enzyme purification was carried out at 4°C with a Bio-Rad BioLogic chromatography system (Bio-Rad, Hercules, Calif.). Cell extracts were applied to a DE-52 anion exchange column equilibrated with 25 mM Tris-HCl buffer (pH 7.2). The column was washed with the same buffer, and protein was eluted with a linear gradient (0 to 0.8 M KCl) in 25 mM Tris-HCl (pH 7.2). Fractions containing toluene cis-dihydrodiol dehydrogenase activity eluted at approximately 0.4 M KCl. These were pooled and concentrated by ultrafiltration with a 30-kDa cut-off membrane filter (Amicon, Danvers, Mass.). The partially purified dehydrogenase preparation, which did not oxidize 3-methylcatechol, was stored at 70°C. The toluene cis-dihydrodiol dehydrogenase (TodD) expressed by IPTG-induced JM109(DE3)(pDTG141-F352V)(pDTG511) was partially purified by an analogous procedure. Protein concentrations were determined by the method of Bradford with bovine serum albumin as standard (10).

cis-Dihydrodiol dehydrogenase assays. Dehydrogenase activities with biphenyl cis-3,4-dihydrodiol were measured spectrophotometrically by following the reduction of NAD⁺ at 340 nm. Since biphenyl cis-2,3-dihydrodiol shows a slight absorbance at 340 nm, activities with this substrate were measured at 350 nm using an extinction coefficient of 5170 M¹ cm¹ for NADH. Reaction mixtures were contained in a final volume of 1.0 ml of 25 mM Tris-HCl (pH 7.2), NAD⁺ (2.6 µmol), and enzyme (0.5 to 1.0 mg of protein). Reactions were started by adding biphenyl cis-dihydrodiol (0.1 to 0.3 µmol).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Activity of modified NDO proteins. The formation of indigo from indole was used to screen for NDO activity. Freshly grown cells of JM109(DE3) carrying modified pDTG141 plasmids were incubated in the presence of indole. Strains producing NDO enzymes with the mutations F352W and F352Y formed white colonies, suggesting that these enzymes were inactive or that indole was no longer a substrate for the modified enzymes. All other NDO variants constructed in this study were active (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   Amino acid substitutions in the  subunit of NDO generated by site-directed mutagenesis

Production of mutant NDO  subunits. Formation of mutant  subunits was demonstrated in Western blots using whole-cell protein samples from induced JM109(DE3) strains expressing the mutant NDO genes. Use of the monoclonal antibody specific for the  subunit of NDO (37) demonstrated that all mutant constructs resulted in the formation of full-length  subunits (Fig. 2). The results show that the inability of NDO variants F352Y and F352W to transform the various substrates (see below) was not due to the absence of protein.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Western blot showing  subunits formed by JM109(DE3) carrying pDTG141 derivatives with the amino acid substitutions indicated below. A monoclonal antibody specific for the  subunit of NDO was used as described in Materials and Methods. Lanes: M, prestained molecular mass markers (Bio-Rad Laboratories); 1, purified wild-type NDO (2 µg); 2, wild-type NDO (pDTG141); 3, vector control (pT7-5); 4, F352G; 5, F352A; 6, F352I; 7, F352T; 8, F352W; 9, F352Y.

Regioselectivity of modified NDO proteins. Biotransformation products were identified by comparison to standards in GC-MS analyses. Biotransformations with naphthalene result in the formation of naphthalene cis-dihydrodiol by wild-type NDO from Pseudomonas sp. NCIB 9816-4 (28) and by JM109(DE3)(pDTG141), which carries the cloned naphthalene dioxygenase genes from NCIB 9816-4 (38). All NDO variants with substitutions at position 352 also formed naphthalene cis-dihydrodiol except the F352Y mutant, which formed no product, and F352W, which formed only a trace amount of naphthalene cis-dihydrodiol.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Regiochemistry and relative product distributions (%) of compounds formed by wild-type and mutant NDO enzymes (A) with biphenyl as substrate and (B) with phenanthrene as substrate. Data for F352V and F352L mutants are from reference 38. Absolute stereochemistry of the biphenyl and phenanthrene dihydrodiols is not intended.

Relative activities of the mutant NDO enzymes. The in vivo rates of formation of naphthalene cis-dihydrodiol by wild-type and mutant NDO enzymes are shown in Table 3. The F352L enzyme produced naphthalene cis-dihydrodiol at wild-type rates, while the F352T, F352V, and F352I mutant enzymes were slightly less efficient, with rates 77 to 83% of wild-type NDO. The F352G and F352A mutant enzymes were the least efficient in catalyzing this reaction. A similar trend is seen in the rates of formation of biphenyl cis-3,4-dihydrodiol from biphenyl by the enzymes with substitutions at position 352 (Table 3). The F352T, V, I, and L mutant enzymes formed biphenyl cis-3,4-dihydrodiol at slightly reduced rates compared to wild-type NDO, while the F352A variant was significantly slower, and rates with the F352G mutant were not measurable. In contrast, all enzymes with substitutions at position 352 were severely defective in forming biphenyl cis-2,3-dihydrodiol from biphenyl (Table 3). These studies demonstrate that the amino acid substitutions at position 352 result in enzymes with a decreased tendency to oxidize at the 2,3 position of biphenyl. Although the regioselectivity was changed in the mutant enzymes, the rates of oxidation at the 3,4 position of biphenyl by the F352T, V, I, and L mutants were similar to that observed with the wild-type enzyme.

Enantioselectivity of modified NDO proteins. The enantiomeric purities of naphthalene cis-dihydrodiol and biphenyl cis-2,3- and cis-3,4-dihydrodiols were determined by chiral stationary-phase high-performance liquid chromatography (HPLC) analysis as described in Materials and Methods. In contrast to wild-type NDO, all enzymes with amino acid substitutions at position 352 formed small amounts of the ()-enantiomer of naphthalene cis-dihydrodiol from naphthalene (Table 4). The enantiomeric composition of biphenyl cis-2,3-dihydrodiol was unaffected by amino acid substitutions at this position, but that of the biphenyl cis-3,4-dihydrodiol was significantly different in all cases from that formed by the wild-type enzyme (Table 4). It is of interest that the NDO variants F352V and F352T formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol to that formed by wild-type NDO (Table 4).

Absolute stereochemistry of biphenyl cis-3,4-dihydrodiol. The optical rotation of the biphenyl cis-3,4-dihydrodiol produced by the F352V mutant was previously reported ([]_D 37.5) (38). The absolute stereochemistry of the ()-biphenyl cis-3,4-dihydrodiol was unequivocally identified as the (3S,4R) enantiomer by preparing the compound through chemoenzymatic synthesis as described in Materials and Methods and outlined in Fig. 1. The synthesis led to the preparation of (+)-biphenyl cis-(3R,4S)-dihydrodiol which was identical in structure but had chiroptical properties opposite to those of the bacterial metabolite (Fig. 4).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   CD spectra of chemoenzymatically derived (+)-biphenyl-cis-(3R,4S)-dihydrodiol (99% enantiomeric purity, bold line), and enzymatically derived ()-biphenyl-cis-(3S,4R)-dihydrodiol (75% enantiomeric purity, dashed line).

Absolute stereochemistry of phenanthrene cis-3,4-dihydrodiol, phenanthrene cis-1,2-dihydrodiol, and anthracene cis-1,2-dihydrodiol. For the phenanthrene cis-1,2-dihydrodiol formed by the F352V mutant of NDO, the methoxy signal of the (S)-MPBA derivative was observed downfield (+72 ppb) from the corresponding signal of the opposite diastereomer formed with (R)-MPBA and predicted an S-configuration at the benzylic center (Table 5). Based on previously documented trends (46), the absolute configuration of the major dihydrodiol formed by the F352V mutant from phenanthrene is phenanthrene cis-(1S,2R)-dihydrodiol (91% enantiomeric purity; 83% relative yield). The facial selectivity in this case was opposite to that shown for wild-type biphenyl dioxygenase from S. yanoikuyae B8/36. Analysis of the (±)-MPBA derivative of the isolated phenanthrene cis-dihydrodiol fraction formed by strain B8/36 showed resolution of the mixed racemates (of 3,4- and 1,2-diols) with minor methoxy signals of the 1,2-diol at 3.148 and 3.220 ppm. The same sample, when derivatized with (S)-MPBA, showed the upfield shifted methoxy signal at 3.148 ppm which corresponds to an R-configuration at the benzylic center, a result consistent with the (1R,2S) configuration that was previously determined (33). The results of the above stereochemical correlation also suggest that the empirical application of the trends in the directional shifts of polycyclic aromatic diols are valid for both the "bay-region" cis-3,4- and "non-bay region" cis-1,2-dihydrodiols of phenanthrene.

Preparation of enantiopure ()-biphenyl cis-(3S,4R)-dihydrodiol and ()-phenanthrene cis-(1S,2R)-dihydrodiol. Toluene cis-dihydrodiol dehydrogenase from P. putida F1 (48) was partially purified to remove 3-methylcatechol 2,3-dioxygenase. This was necessary to eliminate the absorbance at 340 nm by the 3-methylcatechol ring fission product. The partially purified dehydrogenase was examined for its ability to oxidize the enantiomers of biphenyl cis-2,3- and cis-3,4-dihydrodiols. As shown in Table 6, the enzyme specifically oxidized the (+)-enantiomers of both dihydrodiols. Extracts of an E. coli strain carrying pDTG511 (56), which expresses toluene cis-dihydrodiol dehydrogenase from P. putida F1, gave similar results (Table 6). After extraction and analysis by TLC, dihydroxybiphenyl products were detected in all reaction mixtures that contained (+)-biphenyl cis-dihydrodiols. Although a slight amount of NADH was formed in assays with ()-biphenyl 2,3-dihydrodiol (Table 6), we did not detect the formation of any 2,3-dihydroxybiphenyl. On the basis of these findings, a recombinant E. coli strain was constructed which expresses both the P. putida todD gene encoding toluene cis-dihydrodiol dehydrogenase (56) and the NDO F352V mutant enzyme. This strain, JM109(DE3)(pDTG141-F352V)(pDTG511), was used in whole-cell biotransformation experiments. Incubation of this organism with biphenyl as described in Materials and Methods led to the isolation of 32 mg of homochiral ()-biphenyl cis-(3S-4R)-dihydrodiol after purification by PLC. The (+)-enantiomers of biphenyl cis-2,3-dihydrodiol and biphenyl cis-3,4-dihydrodiol were completely converted to their respective catechols, compounds which were easily separated from the ()-biphenyl cis-3,4-dihydrodiol by PLC. The specific rotation []_D 84.4 (c 0.3, methanol), of the ()-biphenyl cis-3,4-dihydrodiol was, with the exception of the sign of rotation, identical to that of the synthetic biphenyl cis-3,4-dihydrodiol. Control E. coli strain JM109(DE3)(pDTG141-F352V)(pDTG512), which contains a truncated todD gene, and strain JM109(DE3)(pDTG141-F352V)(pKT230), which does not contain the todD gene, both formed a 96:4 ratio of biphenyl cis-3,4-dihydrodiol and biphenyl cis-2,3-dihydrodiol.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

NDO has a relaxed substrate specificity that allows the oxidation of more than 70 substrates (45). Previous work has shown that NDO can accept amino acid substitutions at several positions near the active site without losing activity. Most of the resulting enzymes had, at most, minor changes in regioselectivity and no differences in enantioselectivity with the substrates tested (38). In this study, a series of amino acid substitutions at position 352 demonstrated that the amino acid at this position in the  subunit of NDO is critical in determining both the regio- and enantioselectivity of the enzyme with a variety of substrates. In addition, the surrounding amino acids also influence the substrate specificity of NDO. This can be seen by comparing specificities of the NDO mutants F352I and F352T with those of 2-nitrotoluene dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 (3, 35, 36) and 2,4-dinitrotoluene dioxygenase (DNTDO) from Burkholderia sp. strain DNT (53). The deduced amino acid sequences of the  subunits of 2NTDO and DNTDO are 84 and 80% identical to the NDO  subunit, respectively (35, 53). At the position corresponding to Phe-352 in NDO, 2NTDO and DNTDO have an isoleucine and a threonine, respectively. 2NTDO catalyzes the oxidation of 2-nitrotoluene to nitrite and 3-methylcatechol. In a similar reaction, DNTDO converts 2,4-dinitrotoluene to 4-methyl-5-nitrocatechol and nitrite. However, the F352I and F352T mutants of NDO cannot catalyze either reaction (data not shown). In addition, whereas 2NTDO forms 70% (+)-naphthalene cis-(1R,2S)-dihydrodiol, the F352I mutant of NDO forms the cis-dihydrodiol in much higher enantiomeric purity [94% (+)-(1R,2S); Table 4]. The reactions catalyzed by DNTDO and the NDO F352T mutant are similar, producing 96 and 93% (+)-naphthalene cis-(1R,2S)-dihydrodiol, respectively. Biphenyl and phenanthrene are not substrates for 2NTDO and DNTDO (38) but are oxidized by the F352I and F352T mutants of NDO (Fig. 3; Table 3). These results suggest that while the amino acid at position 352 is critical, other amino acids must also play a role in determining substrate specificity in these enzymes.

Introduction of threonine, a smaller and more polar amino acid than phenylalanine, at position 352, resulted in an enzyme with slightly reduced activity (Table 3). However, replacement of Phe-352 with glycine resulted in a defective enzyme, particularly when biphenyl was provided as the substrate (Table 3). Introduction of this small amino acid may have destabilized the protein as suggested in other studies (11). Similarly, substitution with the next smallest amino acid, alanine, resulted in an enzyme with lowered activity. In contrast, hydrophobic amino acids of intermediate size were tolerated at position 352 and resulted in enzymes with good activity (Table 3). Substitution at position 352 by tyrosine or tryptophan, two amino acids that are larger than phenylalanine, resulted in enzymes with little or no measurable activity, suggesting that the active-site pocket is of limited size and cannot tolerate a larger residue at this position.

The asymmetric chemoenzymatic synthesis of (+)-biphenyl cis-(3R,4S)-dihydrodiol (Fig. 1) of known absolute stereochemistry allowed unequivocal assignment of the ()-cis-(3S,4R) configuration to the regioisomer produced by the F352V mutant. The accurate prediction of cis-(3S,4R)-absolute stereochemistry for the same metabolite through analysis of MPBA-esters suggests that the trends observed for these derivatives with a series of polycyclic cis-2,3-dihydrodiols (46) may be applied to cis-3,4-dihydrodiols in the manner described here. Additional data for a series of cis-3,4-dihydrodiols are needed to establish whether this empirical correlation is generally valid. Assignments of absolute stereochemistry for the phenanthrene cis-1,2- and cis-3,4-dihydrodiols and anthracene cis-1,2-dihydrodiol were based on direct comparisons of ¹H-NMR chemical shifts of their MPBA derivatives to those of known MPBA derivatives prepared using biphenyl dioxygenase (Table 5). Distinct chemical shifts allowed the analysis of a mixture of phenanthrene cis-3,4- and cis-1,2-dihydrodiols.

Of the eight amino acid replacements made at position 352 in NDO, the F352V mutant enzyme had an overall substrate specificity that was most different from wild-type NDO. A detailed characterization of the products formed by the F352V mutant demonstrated that the enzyme had the opposite regioselectivity with biphenyl (Fig. 3A) and phenanthrene (Fig. 3B; 38) and a slight change in enantioselectivity with naphthalene (Table 4) and anthracene as substrates (Table 5). In addition, the opposite enantiomers of biphenyl cis-3,4-dihydrodiol and phenanthrene cis-1,2-dihydrodiol were formed in contrast to the enantiomer formed by wild-type NDO (Tables 4 and 5).

Enantioselective diol dehydrogenases have been used to kinetically resolve mixtures of cis-dihydrodiols to form enantiomerically pure compounds (2, 13, 41). Naphthalene-grown whole cells of P. putida NCIMB 8859 selectively oxidized several cis-dihydrodiols, including (+)-naphthalene cis-dihydrodiol, and several substituted benzene dihydrodiols (2). Naphthalene cis-dihydrodiol dehydrogenase purified from P. putida NP exclusively oxidized the (+)-enantiomer of naphthalene cis-dihydrodiol (28). Results from this study indicate that toluene cis-dihydrodiol dehydrogenase from P. putida F1 catalyzes enantioselective reactions with biphenyl cis-2,3-dihydrodiol and biphenyl cis-3,4-dihydrodiol. Only the (+)-enantiomers of each dihydrodiol were oxidized by partially purified enzyme preparations from toluene-induced cells of P. putida F1 and IPTG-induced cells of a recombinant E. coli strain expressing the todD gene (Table 6). Similar enantioselective activities were obtained with partially purified biphenyl and naphthalene cis-dihydrodiol dehydrogenases from Pseudomonas sp. strain LB400 (26) and Pseudomonas sp. strain NCIB 9816-4 (40; data not shown).

The results presented here establish procedures for the production of enantiomers of both biphenyl cis-2,3- and cis-3,4-dihydrodiol (Fig. 5). (+)-Biphenyl cis-(2R,3S)-dihydrodiol is the sole product formed from biphenyl by biphenyl dioxygenase (19, 20), and ()-biphenyl cis-(2S,3R)-dihydrodiol is the major product formed by carbazole dioxygenase (43; Resnick and Gibson, Abstr. 96th Gen. Meet. Am. Soc. Microbiol., 1996, abstr. O-11, p. 355, 1996). The results presented provide methods for the enzymatic or chemoenzymatic synthesis of the (+)- and ()-enantiomers of biphenyl cis-3,4-dihydrodiol. A five-step chemoenzymatic synthesis scheme (Fig. 1) for the formation of (+)-biphenyl cis-(3R,4S)-dihydrodiol utilizes toluene dioxygenase to catalyze the initial step. The combination of the NDO F352V mutant enzyme and toluene cis-dihydrodiol dehydrogenase allowed the formation of ()-biphenyl cis-(3S,4R)-dihydrodiol from biphenyl in one step and required a single purification step. The same strain construction provided a simple method for the enzymatic production of ()-phenanthrene cis-(1S,2R)-dihydrodiol. Formation of these two compounds as sole products in high enantiopurity demonstrates the utility of constructed strains expressing mutated NDO with altered substrate specificity in combination with the regio- and enantioselective resolution afforded by a cis-dihydrodiol dehydrogenase.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Biosynthetic routes to both enantiomers of biphenyl cis-2,3- and cis-3,4-dihydrodiols from biphenyl. BPDO, biphenyl dioxygenases from Pseudomonas sp. LB400 or S. yanoikuyae B8/36 form 100% (+)-biphenyl cis-(2R,3S)-dihydrodiol; CDO; carbazole dioxygenase from P. stutzeri C250 forms >95% ()-biphenyl cis-(2S,3R)-dihydrodiol; TDO, toluene dioxygenase from P. putida UV4 and four synthetic steps (Fig. 1 and Materials and Methods) resulted in 100% (+)-biphenyl cis-(3R,4S)-dihydrodiol; the F352V mutant of NDO and toluene cis-dihydrodiol dehydrogenase (TDD) from P. putida F1, followed by one purification step, resulted in 100% ()-biphenyl cis-(3S,4R)-dihydrodiol.