Desaturation and oxygenation of 1,2-dihydronaphthalene by toluene and naphthalene dioxygenase.

Bacterial strains expressing toluene and naphthalene dioxygenase were used to examine the sequence of reactions involved in the oxidation of 1,2-dihydronaphthalene. Toluene dioxygenase of Pseudomonas putida F39/D oxidizes 1,2-dihydronaphthalene to (+)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene, (+)-(1R)-hydroxy-1,2-dihydronaphthalene, and (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, naphthalene dioxygenase of Pseudomonas sp. strain NCIB 9816/11 oxidizes 1,2-dihydronaphthalene to the opposite enantiomer, (-)-cis-(1R,2S)-dihydroxy-1,2,3,4-tetrahydronaphthalene and the identical (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. Recombinant Escherichia coli strains expressing the structural genes for toluene and naphthalene dioxygenases confirmed the involvement of these enzymes in the reactions catalyzed by strains F39/D and NCIB 9816/11. 1-Hydroxy-1,2-dihydronaphthalene was not formed by strains expressing naphthalene dioxygenase. These results coupled with time course studies and deuterium labelling experiments indicate that, in addition to direct dioxygenation of the olefin, both enzymes have the ability to desaturate (dehydrogenate) 1,2-dihydronaphthalene to naphthalene, which serves as a substrate for cis dihydroxylation.     

Naphthalene dioxygenase: purification and properties of a terminal oxygenase component

Naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 is a multicomponent enzyme system that oxidized naphthalene to cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene. The terminal oxygenase component B was purified to homogeneity by a three-step procedure that utilized ion-exchange and hydrophobic interaction chromatography. The purified enzyme oxidized naphthalene only in the presence of NADH, oxygen, and partially purified preparations of components A and C. An estimated Mr of 158,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of two subunits with molecular weights of ca. 55,000 and 20,000, indicative of an alpha 2 beta 2 quaternary structure. Absorption spectra of the oxidized enzyme showed maxima at 566 (shoulder), 462, and 344 nm, which were replaced by absorption maxima at 520 and 380 nm when the enzyme was reduced anaerobically by stoichiometric quantities of NADH in the presence of the other two components of the naphthalene dioxygenase system. Component B bound naphthalene. Enzyme-bound naphthalene was oxidized to product upon the addition of components A and C, NADH, and O2. These results, together with the detection of the presence of 6.0 g-atoms of iron and 4.0 g-atoms of acid-labile sulfur per mol of the purified enzyme, suggest that component B of the naphthalene dioxygenase system is an iron-sulfur protein which functions in the terminal step of naphthalene oxidation.     

Initial reactions in the oxidation of 1,2-dihydronaphthalene by Sphingomonas yanoikuyae strains.

The substrate oxidation profiles of Sphingomonas yanoikuyae B1 biphenyl-2,3-dioxygenase and cis-biphenyl dihydrodiol dehydrogenase activities were examined with 1,2-dihydronaphthalene and various cis-diols as substrates. m-Xylene-induced cells of strain B1 oxidized 1,2-dihydronaphthalene to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2-3,4-tetrahydronaphthalene as the major product (73% relative yield). Small amounts of (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (15%), naphthalene (6%), and alpha-tetralone (6%) were also formed. Strain B8/36, which lacks an active cis-biphenyl dihydrodiol dehydrogenase, formed (+)-(1R,2S)-cis-1,2-dihydroxy-1,2-dihydronaphthalene (51%), in addition to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene (44%) and (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (5%). The cis-biphenyl dihydrodiol dehydrogenase of strain B1 oxidized both enantiomers of cis-1,2-dihydroxy-1,2-dihydronaphthalene, but only the (+)-(1S,2R)-enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene. The results show that biphenyl dioxygenase expressed by S. yanoikuyae catalyzes dioxygenation, monooxygenation, and desaturation reactions with 1,2-dihydronaphthalene as the substrate, and cis-biphenyl dihydrodiol dehydrogenase catalyzes the enantioselective dehydrogenation of (+)-(1S,2R)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and (+)-(1S,2R)-cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene.     

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.     

Initial reactions in the oxidation of naphthalene by Pseudomonas putida.

A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.     

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.     

Metabolism of naphthalene by cell extracts of Cunninghamella elegans.

Microsomal preparations of Cunninghamella elegans oxidized naphthalene to trans-1,2-dihydroxy-1,2-dihydronaphthalene, 1-naphthol, and 2-naphthol. Enzymatic activity was dependent on the presence of reduced nicotinamide adenine dinucleotide phosphate and oxygen. Reduced microsomal preparations, when treated with carbon monoxide, showed absorption maxima at 450 and 420 nm. The inhibitor 1,2-epoxy-3,3,3-trichloropropane suppressed the formation of trans-1,2-dihydroxy-1,2-dihydronaphthalene and enhanced 1-naphthol formation. The results suggest that the metabolism of naphthalene by fungal microsomes may be analogous to the cytochrome P-450-dependent monooxygenase activity that is associated with mammalian liver microsomes.     

Identification of metabolites from degradation of naphthalene by a Mycobacterium sp.

A Mycobacterium sp. isolated from oil-contaminated sediments was previously shown to mineralize 55% of the added naphthalene to carbon dioxide after 7 days of incubation. In this paper, we report the initial steps of the degradation of naphthalene by a Mycobacterium sp. as determined by isolation of metabolites and incorporation of oxygen from ¹⁸O₂ into the metabolites. The results indicate that naphthalene is initially converted to cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene by dioxygenase and monooxygenase catalyzed reactions, respectively. The ratio of the cis to trans-naphthalene dihydrodiol isomers was approximately 25:1. Thin layer and high pressure liquid chromatographic and mass spectrometric techniques indicated that besides the cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene, minor amounts of ring cleavage products salicylate and catechol were also formed. Thus the formation of both cis and trans-naphthalene dihydrodiols by the Mycobacterium sp. is unique. The down-stream reactions to ring cleavage products proceed through analogous dioxygenase reactions previously reported for the bacterial degradation of naphthalene.     

Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli

The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).     

Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida.

The purification of (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase from cells of Pseudomonas putida grown with toluene as the sole source of carbon and energy is reported. The molecular weight of the enzyme is 104,000 at pH 9.7. The enzyme is composed of four apparently identical subunits with molecular weights of 27,000. The enzyme is specific for nicotinamide adenine dinucleotide and oxidizes a number of cis-dihydrodiols. Both enantiomers of a racemic mixture of cis-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol are oxidized by the enzyme. No enzymatic activity is observed with trans-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol.     

Purification and propeties of (plus)-cis-naphthalene dihydrodiol dehydrogenase of Pseudomonas putida

Cells of Pseudomonas putida, after growth with naphthalene as sole source of carbon and energy, contain an enzyme that oxidizes (+)-cis-1(r),2(s)-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. The purified enzyme has a molecular weight of 102,000 and apparently consists of four 25,500 molecular weight subunits. The enzyme is specific for nicotinamide adenine dinucleotide as an electron acceptor and also oxidizes several other cis-dihydrodiols. However, no enzymatic activity was observed with trans-1,2-dihydronaphthalene, or the K-region cis-dihydrodiols of carcinogenic polycyclic hydrocarbons.     

Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1

Pseudomonas paucimobilis Q1 originally isolated as biphenyl degrading organism (Furukawa et al. 1983), was shown to grow with naphthalene. After growth with biphenyl or naphthalene the strain synthesized the same enzyme for the ring cleavage of 2,3-dihydroxybiphenyl or 1,2-dihydroxynaphthalene. The enzyme, although characterized as 2,3-dihydroxybiphenyl dioxygenase (Taira et al. 1988), exhibited considerably higher relative activity with 1,2-dihydroxynaphthalene. These results demonstrate that this enzyme can function both in the naphthalene and biphenyl degradative pathway.Abbreviations DHBP dihydroxybiphenyl - DHBPDO 2,3-dihydroxybiphenyl dioxygenase - DHDHNDH 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase - DHN 1,2-dihydroxynaphthalene - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBP cis-2-hydroxybenzalpyruvate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - PCB polychlorinated biphenyl - 2NS naphthalene-2-sulfonic acid     

Single turnover chemistry and regulation of O2 activation by the oxygenase component of naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O2, and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the alpha3beta3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe2+ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O2. However, upon reduction of the Rieske cluster and exposure to naphthalene and O2, approximately 0.85 cis-diol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.     

Biotransformation of various substituted aromatic compounds to chiral dihydrodihydroxy derivatives

The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5alpha(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) from Pseudomonas sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the corresponding cis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls were oxidized. The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined. All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene dioxygenases of Pseudomonas putida UV4 and P. putida F39/D. The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

Regio- and stereospecific oxidation of 9,10-dihydroanthracene and 9,10-dihydrophenanthrene by naphthalene dioxygenase: structure and absolute stereochemistry of metabolites.

The oxidation of 9,10-dihydroanthracene and 9,10-dihydrophenanthrene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas putida (NCIB 9816.4. Salicylate-induced cells of P. putida strain 9816/11 and isopropylthiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized 9,10-dihydroanthracene to (+)-cis-1R,2S)-1,2-dihydroxy-1,2,9,10-tetrahydroanthracene (> 95% relative yield; > 95% enantiomeric excess) as the major product. 9-Hydroxy-9,10-dihydroanthracene (< 5% relative yield) was a minor product formed by both organisms. The same cells oxidized 9,10-dihydrophenanthrene to (+)-cis-(3S,4R)-3,4-dihydroxy-3,4,9,10-tetrahydrophenanthrene (70% relative yield; > 95% enantiomeric excess) and (+)-(S)-9-hydroxy-9,10-dihydrophenanthrene (30% relative yield). The major reaction catalyzed by naphthalene dioxygenase with 9,10-dihydroanthracene and 9,10-dihydrophenanthrene was stereospecific dihydroxylation in which both of the previously undescribed cis-diene diols were of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The results suggest that for benzocylic substrates, the location of benzylic carbons influences the type of reaction(s) catalyzed by naphthalene dioxygenase.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

A rapid method for naphtalene dioxygenase assay in whole cells of naphtalene cis-dihydrodiol dehydrogenase blocked Pseudomonas fluoresecens: Screening of potential inducers of dioxygenase activity

A rapid and sensitive photometric method was devised to assay naphthalene dioxygenase in whole cells of Pseudomonas fluorescens NCIMB 40531, a strain derived from a naphthalene-metabolizing isolate by means of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The naphthalene-assimilating pathway of NCIMB 40531 is functionally blocked at the level of cis-1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase and therefore cis-1,2-dihydroxy-1,2-dihydronaphthalene (napthalene dihydrodiol) is accumulated when cultures are supplied with naphthalene.This modified metabolism allowed dioxygenase to be assayed by monitoring product formation. Optimal conditions were selected to give linear optical density vs time curves and reaction rates proportional to dry cell weight (DCW): specific activities of 0.125(+-0.008) mol·min^–1·mgDCW^–1 were consistently obtained in cultures grown on succinate in the presence of naphthalene as inducer. By means of the developed assay, 62 compounds (mainly mono- and bicyclic aromatics) were screened as potential inducers of the dioxygenase activity, when added to the growth medium at the concentration of 100 mg·1^–1: besides naphthalene, the highest activities were induced by 3-methylsalicyclic acid (2-hydroxy-3-methylbenzoic acid), O-acetylsalicylic acid and 5-chlorosalicyclic acid with 0.198, 0.167 and 0.076 mol·min^–1mg DCW^–1, respectively. Under the conditions used, no detectable dioxygenase activity was induced by salicylic acid, which is recognized as the natural inducer of the enzyme in Pseudomonas.     

Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4.

The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp. strain 9816-4. Pseudomonas sp. strain 9816/11 and Escherichia coli JM109(DE3)[pDTG141] oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone. The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol. Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3)[pDTG141]. In addition, indene was identified as an intermediate in indan oxidation. The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol. The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol. The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.