Oxidation of the carcinogens benzo [a] pyrene and benzo [a] anthracene to dihydrodiols by a bacterium.

A mutant strain of Beijerinckia, after growth with succinate plus biphenyl, contains an enzyme system that oxidizes benzo [a] pyrene and benzo [a] anthracene to mixtures of vicinal dihydrodiols. The major dihydrodiol formed from benzo [a] pyrene was identified as cis-9, 10-dihydroxy-9, 10-dihydrobenzo [a] pyrene by comparison with a synthetic sample. Benzo [a] anthracene was metabolized to four dihydrodiols, the major isomer being cis-1, 2-dihydroxy-1, 2-dihydroxy-1, 2-dihydrobenzo [a] anthracene.

Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo.

A fragment of plasmid NAH7 from Pseudomonas putida PpG7 has been cloned and expressed in Escherichia coli HB101. Growth of the recombinant Escherichia coli in nutrient medium results in the formation of indigo. The production of this dye is increased in the presence of tryptophan or indole. Several bacteria that oxidize aromatic hydrocarbons to cis-dihydrodiols also oxidize indole to indigo. The results suggest that indigo formation is due to the combined activities of tryptophanase and naphthalene dioxygenase.

Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase.

BACKGROUND: Pseudomonas sp. NCIB 9816-4 utilizes a multicomponent enzyme system to oxidize naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme component catalyzing this reaction, naphthalene 1,2-dioxygenase (NDO), belongs to a family of aromatic-ring-hydroxylating dioxygenases that oxidize aromatic hydrocarbons and related compounds to cis-arene diols. These enzymes utilize a mononuclear non-heme iron center to catalyze the addition of dioxygen to their respective substrates. The present study was conducted to provide essential structural information necessary for elucidating the mechanism of action of NDO. RESULTS: The three-dimensional structure of NDO has been determined at 2.25 A resolution. The molecule is an alpha 3 beta 3 hexamer. The alpha subunit has a beta-sheet domain that contains a Rieske [2Fe-2S] center and a catalytic domain that has a novel fold dominated by an antiparallel nine-stranded beta-pleated sheet against which helices pack. The active site contains a non-heme ferrous ion coordinated by His208, His213, Asp362 (bidentate) and a water molecule. Asn201 is positioned further away, 3.75 A, at the missing axial position of an octahedron. In the Rieske [2Fe-2S] center, one iron is coordinated by Cys81 and Cys101 and the other by His83 and His104. CONCLUSIONS: The domain structure and iron coordination of the Rieske domain is very similar to that of the cytochrome bc1 domain. The active-site iron center of one of the alpha subunits is directly connected by hydrogen bonds through a single amino acid, Asp205, to the Rieske [2Fe-2S] center in a neighboring alpha subunit. This is likely to be the main route for electron transfer.

Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding.

The three-dimensional structure of the aromatic hydroxylating enzyme naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was recently determined. The refinement of the structure together with cyclic averaging showed that in the active site of the enzyme there is electron density for a flat aromatic compound. This compound appears to be an indole adduct, which in Escherichia coli is derived from tryptophan present in the rich culture medium. An indole-dioxygen adduct has been built which fits the electron density convincingly. Support for this interpretation was obtained from crystals of the enzyme purified from cells grown in the absence of tryptophan which had an empty substrate pocket. These types of crystals were soaked in indole solutions and the position of indole in this complex was similar to the corresponding part in the modelled indole-oxygen adduct. This suggests that a peroxide bound to iron end-on attacks the substrate and forms this intermediate. The substrate position has implications for the substrate specificity of the enzyme. Docking studies with indole, naphthalene and biphenyl inside the substrate pocket of NDO suggest the presence of subpockets where the one close to the active site iron is reserved for the binding of the aromatic ring which is hydroxylated upon catalysis. The plausible location for the binding of dioxygen is between this pocket and the catalytic iron. This is in accordance with the enantiospecificity of the products.

Aromatic hydrocarbon dioxygenases in environmental biotechnology.

Aromatic hydrocarbon dioxygenases belong to a large family of Rieske non-heme iron oxygenases. The dioxygenases have a broad substrate specificity and catalyze enantiospecific reactions with a wide range of substrates. These characteristics make them attractive synthons for the production of industrially and medically important chiral chemicals and also provide essential information for the development of bioremediation technology.

Recombinant Escherichia coli strains synthesize active forms of naphthalene dioxygenase and its individual alpha and beta subunits.

Pseudomonas sp. strain NCIB 9816-4 utilizes naphthalene dioxygenase (NDO), a multicomponent enzyme system, to initiate naphthalene degradation. The terminal component of NDO is an iron-sulfur protein (ISPNAP) with an alpha 2 beta 2 subunit composition. The structural genes encoding the alpha (nahAc) and beta (nahAd) subunits were cloned separately and together into expression vectors where transcription is under the control of the T7 promoter. The recombinant plasmids were transformed into Escherichia coli JM109[pGP1-2] and the synthesis of ISPNAP and its alpha and beta subunits was determined by SDS-PAGE. Low expression of nahAd was shown to be due to inefficient initiation of translation, but a sixfold increase in the amount of beta subunit synthesized was achieved in a coupled translation system. Inclusion bodies were found in all recombinants. Increased levels of soluble active proteins were obtained when E. coli JM109(DE3), used as the host strain for recombinant plasmid, was grown at 25 degrees C. ISPNAP from JM109(DE3)[pDTG121] was purified to homogeneity and shown to have the same properties as those determined for the enzyme purified from NCIB 9816-4. Active ISPNAP was also obtained by mixing cell extracts from separate strains that synthesized the alpha and beta subunits. The availability of large amounts of purified ISPNAP and its alpha and beta subunits will facilitate future studies on the mechanism of oxygen fixation by NDO.

Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1.

The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes. The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation. The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753. The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.

Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. JS42.

The first step in the metabolism of 2-nitrotoluene (2NT) by Pseudomonas sp. JS42 (JS42) is the addition of dioxygen to the aromatic nucleus of 2NT to form 3-methylcatechol with concomitant release of nitrite. This reaction is catalyzed by the three-component dioxygenase system 2-nitrotoluene 2,3-dioxygenase (2NTDO). We report here the cloning and nucleotide (nt) sequence of a 4912-basepair (bp) SacI DNA fragment from JS42 encoding all of the genes required for 2NTDO activity. Sequence analysis of the 4912-bp SacI DNA fragment revealed five open reading frames (ORFs). The amino acid (aa) sequences of the predicted polypeptides from these ORFs exhibit high homology to the aa sequences of polypeptides from other three-component dioxygenase systems. Based on aa sequence analyses, four of the peptides were designated Reductase2NT, Ferredoxin2NT, ISP alpha 2NT and ISP beta 2NT (ISP for iron-sulfur protein) with gene designations ntdAaAbAcAd. The predicted aa sequence from the remaining ORF (ORF2) had identity to ISP alpha subunits from other three-component dioxygenase systems but had a calculated molecular weight (M(r)) of 21,259, which is uncharacteristically small for ISP alpha subunits.

Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1.

The todFC1C2BADE gene cluster in Pseudomonas putida F1 encodes enzymes for the first four steps of toluene degradation, leading to the formation of 2-hydroxypenta-2,4-dienoate (HPD). Here, we report the nucleotide (nt) sequence and expression of the remaining three genes of the tod pathway, downstream from todE and arranged in the order, todGIH. The deduced amino acid (aa) sequences of TodG [HPD hydratase (268 aa)], TodH [4-hydroxy-2-oxovalerate (HO) aldolase (352 aa)] and TodI [acylating aldehyde (AA) dehydrogenase (316 aa)] are compared with the isofunctional proteins present in the meta-cleavage pathways of other bacteria. New sequence motifs are identified. The highly conserved TodH and TodI sequences are potentially useful DNA probes for biomonitoring purposes.

Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4.

The multicomponent enzyme, naphthalene dioxygenase, initiates the metabolism of naphthalene by Pseudomonas putida strains G7 (PpG7) and NCIB 9816-4 (Pp9816-4). The genes involved (nahAaAbAcAd) are encoded by the NAH7 and pDTG1 plasmids, respectively, and form part of the nah operon. The locations of the structural genes were determined on previously cloned fragments of DNA. The nucleotide (nt) sequences were determined for nahAaAb from Pp9816-4 and for nahAaAbAcAd from PpG7. The appropriate open reading frames were identified using N-terminal amino acid sequences determined from the purified proteins. The two nt sequences showed 93% homology, with the least homology seen upstream from the promoter region.

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase.

Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis-dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxgenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis-dihydrodiol.

The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays.

Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.

Fungal oxidation of 3-methylcholanthrene: formation of proximate carcinogenic metabolites of 3-methylcholanthrene.

The filamentous fungus, Cunninghamella elegans, was found to metabolize the potent carcinogen, 3-methylcholanthrene (3-MC) to 1-hydroxy-3-MC, 2-hydroxy-3-MC, 1-keto-3-MC, 2-keto-3-MC and trans-9,10-dihydrodiols of 1-hydroxy-3-MC. In addition several unidentified derivatives of 3-MC were found. The metabolites formed were separated by high pressure liquid chromatography (HPLC) and identified by comparison of retention times, absorbance, fluorescence and mass spectra with those of synthetic standards. Incubation of (+/-)-1-hydroxy-3-MC and (+/-)-2-hydroxy-3-MC with cells of C. elegans indicated that 1-hydroxy-3-MC is metabolized to form diasteromerically related trans-9,10-dihydrodiols of 1-hydroxy-3-MC. Experiments with 3-[14C]MC showed that over a 48-h period, 8.7% of the hydrocarbon was oxidized to organic solvent-soluble metabolic products. Most of the metabolites were polar products, some of which co-chromatographed with trans-9,10-dihydrodiols of 1-hydroxy-3-MC. The results show that C. elegans has the ability to oxidize 3-MC to metabolites that have been implicated as proximate carcinogenic forms of 3-MC in higher organisms.

Beijerinckia sp strain B1: a strain by any other name.

Beijerinckia sp strain B1 grows with biphenyl as its sole source of carbon and energy. A mutant, strain B8/36, oxidized biphenyl to cis-(2S,3R)-dihydroxy-l-phenylcyclohexa-4,6-diene (cis-biphenyl dihydrodiol). Strain B8/36 oxidized anthracene, phenanthrene, benz[a]anthracene and benzo[a]pyrene to cis-dihydrodiols. Other substrates oxidized to cis-dihydrodiols were dibenzofuran, dibenzothiophene and dibenzo-p-dioxin. Biphenyl dioxygenase activity was observed in cells of Beijerinckia B1 and B8/36 after growth in the presence of biphenyl, m-, p-xylene and salicylate. Recent studies have led to the reclassification of Beijerinckia B1 as Sphingomonas yanoikuyae strain B1. Subsequent biotransformation studies showed that S. yanoikuyae B8/36 oxidized chrysene to a bis-cis-diol with hydroxyl substituents at the 3,4- and 9,10-positions. Dihydronaphthalene was oxidized to cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, naphthalene, cis-1,2-dihydroxy-1,2-dihydronaphthalene and 2-hydroxy-1,2-dihydronaphthalene. Anisole and phenetole were oxidized to phenol. Thus the S. yanoikuyae biphenyl dioxygenase catalyzes cis-dihydroxylation, benzylic monohydroxylation, desaturation and dealkylation reactions. To date, the genes encoding biphenyl dioxygenase have not been cloned. However, the nucleotide sequence of a S. yanoikuyaeB1 DNA fragment contains five different alpha subunits as determined by conserved amino acids coordinating iron in a Rieske [2Fe-2S] center and mononuclear iron at the catalytic site. The specific role of the different putative oxygenases in biotransformation reactions catalyzed by S. yanoikuyae is not known and presents an exciting challenge for future studies.

Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1.

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems.

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.

Initial reactions in the bacterial degradation of aromatic hydrocarbons.

Bacteria incorporate two atoms of molecular oxygen into aromatic hydrocarbons with the formation of cis dihydrodiols. The production of cis dihydrodiols has been demonstrated for substrates that range in size from benzene to benzo[a]pyrene. These results are in direct contrast to the mechanisms utilized by mammals, and possibly all eucaryotic organisms, for the oxidation of aromatic hydrocarbons. Thus, mammals incorporate one atom of molecular oxygen into these substrates to form arene oxides. Hydration of the latter compounds produces dihydrodiols that have a trans stereochemistry.

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.