The ferredoxin component of carbazole 1,9a-dioxygenase from N. aromaticivorans IC177 was crystallized and diffraction data were collected to 2.0 Å resolution.
Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals, which were improved by macroseeding, diffract to 2.0 Å resolution and belong to space group P41212.
ferredoxins; carbazole; Rieske nonhaem iron oxygenase system; Rieske-type proteins
The electron-transfer complex between the terminal oxygenase and ferredoxin of carbazole 1,9a-dioxygenase was crystallized and diffraction data were collected to 1.90 Å resolution.
Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 Å and belongs to space group P21, with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 Å, α = γ = 90, β = 103.8°. Diffraction data for the type I crystal gave an overall R
merge of 8.0% and a completeness of 100%. Its V
M value is 2.63 Å3 Da−1, indicating a solvent content of 53.2%.
angular dioxygenases; carbazole; electron-transfer complexes; Rieske non-haem iron oxygenase systems; Rieske-type ferredoxins; Rieske-type proteins
The NAD(P)H:ferredoxin oxidoreductase in carbazole 1,9a-dioxygenase from Janthinobacterium sp. J3 was crystallized and diffraction data were collected to 2.60 Å resolution.
Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 Å and belonged to space group P42212, with unit-cell parameters a = b = 158.7, c = 81.4 Å. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 Å resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 Å.
angular dioxygenases; NAD(P)H:ferredoxin oxidoreductases; Rieske nonhaem iron oxygenase system; electron transfer; carbazole
The ferredoxin component of carbazole 1,9a-dioxygenase (CARDO-F) is involved in an electron-transfer reaction. The CARDO-F from Novosphingobium sp. KA1 was crystallized under anaerobic conditions and diffracted to a resolution of 1.9 Å.
Novosphingobium sp. KA1 uses carbazole 1,9a-dioxygenase (CARDO) as the first dioxygenase in its carbazole-degradation pathway. The CARDO of KA1 contains a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. In contrast to the CARDO systems of other species, the ferredoxin component of KA1 is a putidaredoxin-type protein. This novel ferredoxin was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals belong to space group C2221 and diffraction data were collected to a resolution of 1.9 Å (the diffraction limit was 1.6 Å).
carbazole; putidaredoxin-type proteins; Rieske nonhaem iron oxygenases
The terminal oxygenase component (Oxy) of carbazole 1,9a-dioxygenase (CARDO) catalyzes dihydroxylation of the aromatic ring. The Oxy of CARDO from Novosphingobium sp. KA1 was crystallized and the crystals diffracted to a resolution of 2.1 Å.
Carbazole 1,9a-dioxygenase (CARDO) is the initial dioxygenase in the carbazole-degradation pathway of Novosphingobium sp. KA1. The CARDO from KA1 consists of a terminal oxygenase (Oxy), a putidaredoxin-type ferredoxin and a ferredoxin reductase. The Oxy from Novosphingobium sp. KA1 was crystallized at 277 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction data were collected to a resolution of 2.1 Å. The crystals belonged to the monoclinic space group P21. Self-rotation function analysis suggested that the asymmetric unit contained two Oxy trimers; the Matthews coefficient and solvent content were calculated to be 5.9 Å3 Da−1 and 79.1%, respectively.
carbazole; Novosphingobium; Rieske nonhaem iron oxygenases; sphingomonads; terminal oxygenases
The ferredoxin reductase component of carbazole 1,9a-dioxygenase (Red) is involved in electron transfer from NAD(P)H to ferredoxin. The class IIA Red from Novosphingobium sp. KA1 was crystallized and the crystal diffracted to a resolution of 1.58 Å.
Carbazole 1,9a-dioxygenase (CARDO) is the initial enzyme of the carbazole-degradation pathway. The CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase (Red) and is classified as a class IIA Rieske oxygenase. Red from KA1 was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG 4000. The crystal diffracted to 1.58 Å resolution and belonged to space group P32, with unit-cell parameters a = b = 92.2, c = 78.6 Å, α = γ = 90, β = 120°. Preliminary analysis of the X-ray diffraction data revealed that the asymmetric unit contained two Red monomers. The crystal appeared to be a merohedral twin, with a twin fraction of 0.32 and twin law (−h, −k, l).
carbazole; Rieske nonhaem iron oxygenases; ferredoxin reductases
Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex.
In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding.
The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O–O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.
A crystal was obtained of the complex between reduced terminal oxygenase and oxidized ferredoxin components of carbazole 1,9a-dioxygenase. The crystal belonged to space group P21 and diffracted to 2.25 Å resolution.
The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Å and belonged to space group P21, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 Å, α = γ = 90, β = 100.1°. The V
M value is 2.85 Å3 Da−1, indicating a solvent content of 56.8%.
Rieske nonhaem iron oxygenase; electron-transfer complex; terminal oxygenase; ferredoxin; carbazole 1,9a-dioxygenase
Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.
Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.
The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.
Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.
Carbazole is a recalcitrant compound with a dioxin-like structure and possesses mutagenic and toxic activities. Bacteria respond to a xenobiotic by recruiting exogenous genes to establish a pathway to degrade the xenobiotic, which is necessary for their adaptation and survival. Usually, this process is mediated by mobile genetic elements such as plasmids, transposons, and insertion sequences.
The genes encoding the enzymes responsible for the degradation of carbazole to catechol via anthranilate were cloned, sequenced, and characterized from a carbazole-degrading Sphingomonas sp. strain XLDN2-5. The car gene cluster (carRAaBaBbCAc) and fdr gene were accompanied on both sides by two copies of IS6100 elements, and organized as IS6100::ISSsp1-ORF1-carRAaBaBbCAc-ORF8-IS6100-fdr-IS6100. Carbazole was converted by carbazole 1,9a-dioxygenase (CARDO, CarAaAcFdr), meta-cleavage enzyme (CarBaBb), and hydrolase (CarC) to anthranilate and 2-hydroxypenta-2,4-dienoate. The fdr gene encoded a novel ferredoxin reductase whose absence resulted in lower transformation activity of carbazole by CarAa and CarAc. The ant gene cluster (antRAcAdAbAa) which was involved in the conversion of anthranilate to catechol was also sandwiched between two IS6100 elements as IS6100-antRAcAdAbAa-IS6100. Anthranilate 1,2-dioxygenase (ANTDO) was composed of a reductase (AntAa), a ferredoxin (AntAb), and a two-subunit terminal oxygenase (AntAcAd). Reverse transcription-PCR results suggested that carAaBaBbCAc gene cluster, fdr, and antRAcAdAbAa gene cluster were induced when strain XLDN2-5 was exposed to carbazole. Expression of both CARDO and ANTDO in Escherichia coli required the presence of the natural reductases for full enzymatic activity.
We predict that IS6100 might play an important role in the establishment of carbazole-degrading pathway, which endows the host to adapt to novel compounds in the environment. The organization of the car and ant genes in strain XLDN2-5 was unique, which showed strong evolutionary trail of gene recruitment mediated by IS6100 and presented a remarkable example of rearrangements and pathway establishments.
The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the kcat/Km for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.
All three components of the toluene dioxygenase system have been expressed, purified and crystallized.
Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme system consisting of a reductase (ReductaseTOL), a ferredoxin (FerredoxinTOL) and a Rieske non-heme iron dioxygenase (OxygenaseTOL). The three components and the apoenzyme of the dioxygenase (apo-OxygenaseTOL) were overexpressed, purified and crystallized. ReductaseTOL diffracts to 1.8 Å and belongs to space group P41212, with unit-cell parameters a = b = 77.1, c = 156.3 Å. FerredoxinTOL diffracts to 1.2 Å and belongs to space group P21, with unit-cell parameters a = 30.5, b = 52.0, c = 30.95 Å, β = 113.7°. Apo-OxygenaseTOL and OxygenaseTOL diffract to 3.2 Å and belong to space group P4332, with unit-cell parameters a = 235.9 Å and a = 234.5 Å, respectively.
toluene 2,3-dioxygenase enzyme system
Carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-p-dioxin. It was revealed by gas chromatography-mass spectrometry and 1H and 13C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2′,3-trihydroxydiphenylmethane and 2,2′,3-trihydroxydiphenyl sulfide, respectively. Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds. In addition to the angular dioxygenation, CARDO catalyzed the cis dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl. Naphthalene and biphenyl were converted by CARDO to cis-1,2-dihydroxy-1,2-dihydronaphthalene and cis-2,3-dihydroxy-2,3-dihydrobiphenyl, respectively. On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively. These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation, cis dihydroxylation, and monooxygenation. The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO.
In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.
The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene.
In this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted.
This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.
Anthranilate (2-aminobenzoate) is an important intermediate in tryptophan metabolism. In order to investigate the degradation of tryptophan through anthranilate by Burkholderia cepacia, several plasposon mutations were constructed of strain DBO1 and one mutant with the plasposon insertion in the anthranilate dioxygenase (AntDO) genes was chosen for further study. The gene sequence obtained from flanking DNA of the plasposon insertion site revealed unexpected information. B. cepacia DBO1 AntDO (designated AntDO-3C) is a three-component Rieske-type [2Fe-2S] dioxygenase composed of a reductase (AndAa), a ferredoxin (AndAb), and a two-subunit oxygenase (AndAcAd). This is in contrast to the two-component (an oxygenase and a reductase) AntDO enzyme from Acinetobacter sp. strain ADP1, P. aeruginosa PAO1, and P. putida P111. AntDO from strains ADP1, PAO1, and P111 are closely related to benzoate dioxygenase, while AntDO-3C is closely related to aromatic hydrocarbon dioxygenases from Novosphingobium aromaticivorans F199 and Sphingomonas yanoikuyae B1 and 2-chlorobenzoate dioxygenase from P. aeruginosa strains 142 and JB2. Escherichia coli cells expressing the functional AntDO-3C genes transform anthranilate and salicylate (but not 2-chlorobenzoate) to catechol. The enzyme includes a novel reductase whose absence results in less efficient transformation of anthranilate by the oxygenase and ferredoxin. AndR, a possible AraC/XylS-type transcriptional regulator, was shown to positively regulate expression of the andAcAdAbAa genes. Anthranilate was the only effector (of 12 aromatic compounds tested) that was able to induce expression of the genes.
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 α3β3 hexamer. The apparent Km of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM−1 min−1 for 2NT and 1.2 μM−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.
We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.
Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.
Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the α and β subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9.2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the α subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11,12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the α subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.
The crystal structures of the three-component toluene 2,3-dioxygenase system provide a model for electron transfer among bacterial Rieske non-heme iron dioxygenases.
Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe–2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe–2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
toluene; dioxygenases; electron transfer; Rieske clusters; reductases; ferredoxins; NADH; FAD
The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.