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1.  Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase 
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%.
doi:10.1107/S1744309105014557
PMCID: PMC1952320  PMID: 16511100
angular dioxygenases; carbazole; electron-transfer complexes; Rieske non-haem iron oxygenase systems; Rieske-type ferredoxins; Rieske-type proteins
2.  Crystallization and preliminary X-ray diffraction studies of a terminal oxygenase of carbazole 1,9a-­dioxygenase from Novosphingobium sp. KA1 
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.
doi:10.1107/S1744309110034949
PMCID: PMC3001653  PMID: 21045300
carbazole; Novosphingobium; Rieske nonhaem iron oxygenases; sphingomonads; terminal oxygenases
3.  Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase component of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1 
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).
doi:10.1107/S1744309110014491
PMCID: PMC2882777  PMID: 20516607
carbazole; Rieske nonhaem iron oxygenases; ferredoxin reductases
4.  Crystallization and preliminary X-ray diffraction studies of a novel ferredoxin involved in the dioxygenation of carbazole by Novosphingobium sp. KA1 
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).
doi:10.1107/S1744309108016278
PMCID: PMC2443972  PMID: 18607094
carbazole; putidaredoxin-type proteins; Rieske nonhaem iron oxygenases
5.  Crystallization and preliminary crystallographic analysis of the ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 
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.
doi:10.1107/S1744309107041437
PMCID: PMC2339720  PMID: 17909288
ferredoxins; carbazole; Rieske nonhaem iron oxygenase system; Rieske-type proteins
6.  Crystallization and preliminary X-ray diffraction studies of the terminal oxygenase component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 
The terminal oxygenase component of carbazole 1,9a-dioxygenase from N. aromaticivorans IC177 was crystallized and diffraction data were collected to 2.30 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 terminal oxygenase component (43.9 kDa) of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 8000 as the precipitant. The crystals diffract to 2.3 Å resolution and belong to space group C2.
doi:10.1107/S1744309106044939
PMCID: PMC2225353  PMID: 17142899
angular dioxygenases; carbazole; Rieske nonhaem iron oxygenase system; Rieske-type protein
7.  Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1472-6807-12-15
PMCID: PMC3423008  PMID: 22727022
8.  Plasmid pCAR3 Contains Multiple Gene Sets Involved in the Conversion of Carbazole to Anthranilate†  
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.
doi:10.1128/AEM.72.5.3198-3205.2006
PMCID: PMC1472349  PMID: 16672458
9.  Purification and Characterization of Carbazole 1,9a-Dioxygenase, a Three-Component Dioxygenase System of Pseudomonas resinovorans Strain CA10 
Applied and Environmental Microbiology  2002;68(12):5882-5890.
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.
doi:10.1128/AEM.68.12.5882-5890.2002
PMCID: PMC134387  PMID: 12450807
10.  Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10. 
Journal of Bacteriology  1997;179(15):4850-4858.
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.
PMCID: PMC179333  PMID: 9244274
11.  Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177†  
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.
doi:10.1128/AEM.72.5.3321-3329.2006
PMCID: PMC1472339  PMID: 16672473
12.  Purification, crystallization and preliminary X-ray diffraction studies of the three components of the toluene 2,3-dioxygenase enzyme system 
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.
doi:10.1107/S1744309105017549
PMCID: PMC1952458  PMID: 16511124
toluene 2,3-dioxygenase enzyme system
13.  The Genes Coding for the Conversion of Carbazole to Catechol Are Flanked by IS6100 Elements in Sphingomonas sp. Strain XLDN2-5 
PLoS ONE  2010;5(4):e10018.
Background
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.
Findings
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0010018
PMCID: PMC2848856  PMID: 20368802
14.  Crystallization and preliminary X-ray analysis of the Rieske-type [2Fe–2S] ferredoxin component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102 
BphA3, a Rieske-type [2Fe–2S] ferredoxin, was crystallized by the hanging-drop vapour-diffusion method. A molecular-replacement calculation yielded a satisfactory solution.
BphA3, a Rieske-type [2Fe–2S] ferredoxin component of a biphenyl dioxygenase (BphA) from Pseudomonas sp. strain KKS102, was crystallized by the hanging-drop vapour-diffusion method. Two crystal forms were obtained from the same conditions. The form I crystal belongs to space group P21212, with unit-cell parameters a = 26.3, b = 144.3, c = 61.5 Å, and diffracted to 2.45 Å resolution. The form II crystal belongs to space group P212121, with unit-cell parameters a = 26.2, b = 121.3, c = 142.7 Å, and diffracted to 2.8 Å resolution. A molecular-replacement calculation using BphF as a search model yielded a satisfactory solution for both forms.
doi:10.1107/S1744309106017799
PMCID: PMC2243079  PMID: 16754990
ferredoxins; electron transfer; Rieske-type [2Fe–2S] clusters
15.  Crystallization and preliminary X-ray analysis of the reduced Rieske-type [2Fe–2S] ferredoxin derived from Pseudomonas sp. strain KKS102 
The reduced form of BphA3, a Rieske-type [2Fe–2S] ferredoxin, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions. A molecular-replacement calculation yielded a satisfactory solution.
The reduced form of BphA3, a Rieske-type [2Fe–2S] ferredoxin component of the biphenyl dioxygenase BphA from Pseudomonas sp. strain KKS102, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions. The crystal belongs to space group P3121, with unit-cell parameters a = b = 49.6, c = 171.9 Å, and diffracts to a resolution of 1.95 Å. A molecular-replacement calculation using oxidized BphA3 as a search model yielded a satisfactory solution.
doi:10.1107/S1744309107009992
PMCID: PMC2330217  PMID: 17401203
ferredoxins; electron transfer; Rieske-type [2Fe–2S] cluster; reduced form; anaerobic conditions
16.  Crystallization and preliminary X-ray analysis of the electron-transfer complex of Rieske-type [2Fe–2S] ferredoxin and NADH-dependent ferredoxin reductase derived from Acidovorax sp. strain KKS102 
The electron-transfer complex of BphA3, a Rieske-type [2Fe–2S] ferredoxin, and BphA4, a NADH-dependent ferredoxin reductase, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions.
The electron-transfer complex of BphA3, a Rieske-type [2Fe–2S] ferredoxin, and BphA4, a NADH-dependent ferredoxin reductase, was crystallized using the sitting-drop vapour-diffusion method under anaerobic conditions. The obtained crystals were analyzed by SDS–PAGE, which showed that they contained both BphA3 and BphA4. The crystals belong to space group P21, with unit-cell parameters a = 60.60, b = 173.72, c = 60.98 Å, β = 115.8°, and diffracted to a resolution of 1.9 Å.
doi:10.1107/S1744309107023007
PMCID: PMC2335082  PMID: 17554177
electron-transfer complexes; ferredoxins; NADH-dependent ferredoxin reductases; anaerobic conditions
17.  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 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.
doi:10.1128/AEM.71.7.3806-3814.2005
PMCID: PMC1169052  PMID: 16000792
18.  Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase from Rhodopseudomonas palustris CGA009 
Palustrisredoxin reductase (RPA3782, PuR), a flavin-dependent ferredoxin reductase, is an essential component of the Class I cytochrome P450 systems in Rhodopseudomonas palustris CGA009. Crystals of PuR that diffract to 2.2 Å resolution have been obtained.
Palustrisredoxin reductase from Rhodopseudomonas palustris CGA009, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (ONFR) family, catalyzes electron transfer from NADH to ferredoxins. It is an essential component of the cytochrome P450 systems in R. palustris CGA009, a model organism with diverse metabolic pathways. Here, the crystallization of palustrisredoxin reductase is reported. The crystals belong to the trigonal space group P3221, with unit-cell parameters a = 107.5, b = 107.5, c = 69.9 Å, and diffract to 2.2 Å resolution on a synchrotron source.
doi:10.1107/S1744309107017411
PMCID: PMC2335014  PMID: 17565187
palustrisredoxin reductase; ferredoxin reductases
19.  Diverse Oxygenations Catalyzed by Carbazole 1,9a-Dioxygenase from Pseudomonas sp. Strain CA10 
Journal of Bacteriology  1999;181(10):3105-3113.
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.
PMCID: PMC93765  PMID: 10322011
20.  Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system 
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.
doi:10.1107/S0907444908036524
PMCID: PMC2628974  PMID: 19153463
toluene; dioxygenases; electron transfer; Rieske clusters; reductases; ferredoxins; NADH; FAD
21.  Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1 
A hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from S. solfataricus P1 has been crystallized as a recombinant protein with a vector-derived long N-terminal extension region. The P43212 crystals of recombinant ARF diffracted to 1.85 Å resolution using synchrotron radiation.
The hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe–2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-­terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 Å resolution and belonged to the tetragonal space group P43212, with unit-cell parameters a = 60.72, c = 83.31 Å. The asymmetric unit contains one protein molecule.
doi:10.1107/S1744309110019263
PMCID: PMC2898476  PMID: 20606288
Rieske-type ferredoxins; [2Fe–2S] clusters; archaea; Sulfolobus solfataricus
22.  A Novel Aromatic-Ring-Hydroxylating Dioxygenase from the Diterpenoid-Degrading Bacterium Pseudomonas abietaniphila BKME-9 
Journal of Bacteriology  1999;181(9):2675-2682.
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.
PMCID: PMC93704  PMID: 10217753
23.  Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1472-6807-7-10
PMCID: PMC1847435  PMID: 17349044
24.  Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a novel plant-type ferredoxin/thioredoxin reductase-like protein from Methanosarcina acetivorans  
A novel ferredoxin/thioredoxin reductase-like protein from M. acetivorans was heterologously expressed in E. coli, purified and then subjected to crystallization. Preliminary X-ray diffraction studies revealed that the crystal belonged to a primitive cubic space group, with unit-cell parameters a = b = c = 92.72 Å.
The genome of Methanosarcina acetivorans contains a gene (ma1659) that is predicted to encode an uncharacterized chimeric protein containing a plant-type ferredoxin/thioredoxin reductase-like catalytic domain in the N-terminal region and a bacterial-like rubredoxin domain in the C-terminal region. To understand the structural and functional properties of the protein, the ma1659 gene was cloned and overexpressed in Escherichia coli. Crystals of the MA1659 protein were grown by the sitting-drop method using 2 M ammonium sulfate, 0.1 M HEPES buffer pH 7.5 and 0.1 M urea. Diffraction data were collected to 2.8 Å resolution using the remote data-collection feature of the Advanced Light Source, Lawrence Berkeley National Laboratory. The crystal belonged to the primitive cubic space group P23 or P213, with unit-cell parameters a = b = c = 92.72 Å. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient (V M) of 3.55 Å3 Da−1, corresponding to a solvent content of 65%.
doi:10.1107/S1744309111017234
PMCID: PMC3144793  PMID: 21795791
ferredoxin/thioredoxin reductase-like proteins; rubredoxin; Methanosarcina acetivorans; archaea
25.  X-ray structure of a soluble Rieske-type ferredoxin from Mus musculus  
The X-ray crystal structure of a soluble Rieske ferredoxin from M. musculus was solved at 2.07 Å resolution, revealing an iron–sulfur cluster-binding domain with similar architecture to the Rieske-type domains of bacterial aromatic dioxygenases. The ferredoxin was also shown to be capable of accepting electrons from both eukaryotic and prokaryotic oxidoreductases.
The 2.07 Å resolution X-ray crystal structure of a soluble Rieske-type ferredoxin from Mus musculus encoded by the gene Mm.266515 is reported. Although they are present as covalent domains in eukaryotic membrane oxidase complexes, soluble Rieske-type ferredoxins have not previously been observed in eukaryotes. The overall structure of the mouse Rieske-type ferredoxin is typical of this class of iron–sulfur proteins and consists of a larger partial β-barrel domain and a smaller domain containing Cys57, His59, Cys80 and His83 that binds the [2Fe–2S] cluster. The S atoms of the cluster are hydrogen-bonded by six backbone amide N atoms in a pattern typical of membrane-bound high-potential eukaryotic respiratory Rieske ferredoxins. However, phylogenetic analysis suggested that the mouse Rieske-type ferredoxin was more closely related to bacterial Rieske-type ferredoxins. Correspondingly, the structure revealed an extended loop most similar to that seen in Rieske-type ferredoxin subunits of bacterial aromatic dioxygenases, including the positioning of an aromatic side chain (Tyr85) between this loop and the [2Fe–2S] cluster. The mouse Rieske-type ferredoxin was shown to be capable of accepting electrons from both eukaryotic and prokaryotic oxidoreductases, although it was unable to serve as an electron donor for a bacterial monooxygenase complex. The human homolog of mouse Rieske-type ferredoxin was also cloned and purified. It behaved identically to mouse Rieske-type ferredoxin in all biochemical characterizations but did not crystallize. Based on its high sequence identity, the structure of the human homolog is likely to be modeled well by the mouse Rieske-type ferredoxin structure.
doi:10.1107/S0907444908021653
PMCID: PMC2631127  PMID: 18703841
Rieske-type ferredoxins; iron–sulfur proteins; oxidoreductases

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