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Pseudomonas sp. strain 9816-4 grows with naphthalene as the sole source of carbon and energy (9). The initial reaction is catalyzed by a multicomponent enzyme system designated naphthalene dioxygenase (NDO) (11,12,23,24). NDO catalyzes the NAD(P)H-dependent enantiospecific incorporation of dioxygen into naphthalene to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (26, 27) ( Fig. 1). An analogous reaction is catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1, where enantiomerically pure (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) is the first detectable oxidation product (17,31,60). TDO also catalyzes the enantiospecific oxidation of naphthalene to (ϩ)-cis-naphthalene dihydrodiol (18,39).In addition to the enantiospecific oxidation of naphthalene and toluene, NDO and TDO from the above strains oxidize many related aromatic compounds to optically active dihydrodiols (10,18,28,30). Other bacterial dioxygenases show similar properties, and more than 130 chiral arene cis-dihydrodiols have been produced from a small number of strains (7,35,48). The high enantiomeric purity of these compounds has led to their use as chiral synthons in the enantiospecific synthesis of a wide variety of biologically active natural products (7,8,46,57). The present studies focus on another facet of this interesting group of dioxygenases, that is, their ability to catalyze reactions other than the formation of arene cis-dihydrodiols. For example, the TDO expressed by P. putida F39/D oxidizes indan to (1R)-indanol and oxidizes indene to cis-(1S,2R)-indandiol and (1S)-indenol (55). Similar reactions have been reported for TDO from P. putida UV4, although the 1-indenol produced by this strain is the (1R)-enantiomer (3, 5).We now report the identification and absolute stereochemistry of the products formed from indan and indene by NDO from Pseudomonas sp. strain 9816-4 and confirm earlier observations on the desaturation of indan to indene by NDO (22). MATERIALS AND METHODSOrganisms. Pseudomonas sp. strain 9816/11 is a mutant which oxidizes naphthalene stoichiometrically to (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (40). This organism is a derivative of Pseudomonas sp. strain 9816-4 (9, 59), which harbors the genes for naphthalene catabolism on an 83-kb NAH plasmid designated pDTG1 (45). Pseudomonas sp. strain 9816/C84, a cured strain, was used as a control in experiments with strain 9816/11. Escherichia coli strain JM109 (DE3)[pDTG141] contains the structural genes (nahAaAbAcAd) for NDO in plasmid pT7-5 (50). Expression of NDO in this strain is inducible by the addition of isopropylthiogalactopyranoside (IPTG). E. coli JM109(DE3)[pT7-5] was used as a control in experiments with strain JM109(DE3) [pDTG141].Biotransformation experiments. Strain 9816/11 was grown at 30ЊC in mineral salts basal medium (MSB) (49) with 0.2% (wt/vol) pyruvate as a carbon source in the presence of 0.05% (wt/vol) salicylate or anthranilate. These aromatic acids induce the s...

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 adds both atoms of the dioxygen molecule to styrene to form (R)-1-phenyl-1,2-ethanediol. Product formation is tightly coupled to dioxygen consumption and NADH oxidation. NDO oxidizes styrene-d 8 at almost the same initial rate as styrene. The results indicate that dioxygen activation by NDO is different from that by cytochrome P-450 and other monooxygenases, which oxidize styrene to styrene 1,2-oxide.Bacterial dioxygenases which contain non-heme iron and Rieske-type (2Fe-2S) redox clusters play a crucial role in the initiation of the degradation of many aromatic hydrocarbons. These enzymes add both atoms of the dioxygen molecule to the aromatic ring of the substrate to form cis-dihydrodiols (10). One well-known example is the three-component naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 (6, 7, 11, 12, 26, 27), which catalyzes the homochiral dihydroxylation of naphthalene to (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) in the presence of dioxygen and NAD(P)H (15, 16) ( Fig. 1). Recent studies have shown that NDO exhibits a relaxed substrate specificity and catalyzes multiple oxidative reactions which lead to stereospecific monohydroxylation (9, 24, 31), desaturation (9, 23, 29), stereospecific sulfoxidation (18), O dealkylation (23), and N dealkylation (19) with appropriate substrates.Almost all of the reactions catalyzed by NDO are also catalyzed by cytochrome P-450 (hereafter called P-450). However, P-450 has not been reported to form cis-dihydrodiols and NDO has not been reported to form epoxides or catalyze National Institutes of Health shift reactions (5). We used styrene as a substrate for NDO to probe for epoxide formation, since P-450 monooxygenases are known to oxidize styrene to styrene 1,2-oxide (8, 34).Identification of reaction products. The oxidation of styrene by purified NDO components (reductase NAP , ferredoxin NAP , and ISP NAP ) (19, 28) was conducted in 2 ml of 50 mM sodium 2-(N-morpholino)ethanesulfonate (MES) buffer, pH 6.8, containing NADH (0.5 mol), Reductase NAP (16 g), Ferredoxin NAP (35 g), ISP NAP (50 g), Fe(NH 4 ) 2 (SO 4 ) 2 ⅐ 6H 2 O (0.1 mol), and styrene (0.25 mol). After 2 h, an internal standard (25 l of a 10 mM methanol stock solution of 1-phenethyl alcohol) was added to the reaction mixture, which was then extracted with ethyl acetate, concentrated, and analyzed by gas chromatography-mass spectrometry as described previously (24). Only one product, which eluted at 10.72 min and gave a mass spectrum identical to that given by authentic 1-phenyl-1,2-ethanediol, was detected. This product was purified by thin-layer chromatography (solvent, chloroform-acetone ϭ 8:2, R f ϭ 0.19), and its enantiomeric composition was determined by chiral stationary-phase-high-performance liquid chromatography (HPLC) on a Chiralcel OB-H column (4.6 mm by 25 cm, 5-m particle size; Chiral Technologies Inc., Exton, Pa.) (24). Enantiomers were eluted with hexane and 2-propanol ...

Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18 O 2 , H 2 18 O, and D 2 O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.