Terminal Oxygenase Component Research Articles

Methods used to determine the structure of the oxygenase component of naphthalene 1,2 dioxygenase.

Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.

A Protochlorophyllide (Pchlide) a Oxygenase for Plant Viability

Higher plants contain a small, 5-member family of Rieske non-heme oxygenases that comprise the inner plastid envelope protein TIC55, phaeophorbide a oxygenasee (PAO), chlorophyllide a oxygenase (CAO), choline monooxygenase, and a 52 kDa protein (PTC52) associated with the precursor NADPH:protochlorophyllide (Pchlide) oxidoreductase A (pPORA) A translocon (PTC). Some of these chloroplast proteins have documented roles in chlorophyll biosynthesis (CAO) and degradation (PAO and TIC55), whereas the function of PTC52 remains unresolved. Biochemical evidence provided here identifies PTC52 as Pchlide a oxygenase of the inner plastid envelope linking Pchlide b synthesis to pPORA import. Protochlorophyllide b is the preferred substrate of PORA and its lack no longer allows pPORA import. The Pchlide b-dependent import pathway of pPORA thus operates in etiolated seedlings and is switched off during greening. Using dexamethasone-induced RNA interference (RNAi) we tested if PTC52 is involved in controlling both, pPORA import and Pchlide homeostasis in planta. As shown here, RNAi plants deprived of PTC52 transcript and PTC52 protein were unable to import pPORA and died as a result of excess Pchlide a accumulation causing singlet oxygen formation during greening. In genetic studies, no homozygous ptc52 knock-out mutants could be obtained presumably as a result of embryo lethality, suggesting a role for PTC52 in the initial greening of plant embryos. Phylogenetic studies identified PTC52-like genes amongst unicellular photosynthetic bacteria and higher plants, suggesting that the biochemical function associated with PTC52 may have an ancient evolutionary origin. PTC52 also harbors conserved motifs with bacterial oxygenases such as the terminal oxygenase component of 3-ketosteroid 9-alpha-hydroxylase (KshA) from Rhodococcus rhodochrous. 3D-modeling of PTC52 structure permitted the prediction of amino acid residues that contribute to the substrate specificity of this enzyme. In vitro-mutagenesis was used to test the predicted PTC52 model and provide insights into the reaction mechanism of this Rieske non-heme oxygenase.

Degradation of three monochlorobenzoate isomers by different bacteria isolated from a contaminated soil

Three monochlorobenzoate isomer-utilizing bacterial strains, Pseudomonas sp. 2-CBA, Pseudomonas sp. 3-CBA, and Hydrogenophaga sp. 4-CBA, were isolated from a haloaromatic-contaminated soil in a disused chemical factory in Nanjing, China. Compared to strain 3-CBA and strain 4-CBA, which could only utilize its corresponding monochlorobenzoate isomer (3-chlorobenzoate and 4-chlorobenzoate) as the sole carbon source for growth, strain 2-CBA could utilize both 2-chlorobenzoate and 3-chlorobenzoate for growth. The three monochlorobenzoate isomers were catabolized via three different dechlorination pathways in the three isolates, respectively. 3-Chlorobenzoate was initially catabolized by the benzoate 1,2-dioxygenase (BenABCD) in strain 3-CBA, whereas 2-chlorobenzoate was catabolized by the hybrid 2-halobenzoate 1,2-dioxygenase system in strain 2-CBA, which comprised of the terminal oxygenase component of 2-halobenzoate dioxygenase system (CbdAB) and the reductase component of benzoate 1,2-dioxygenase system (BenC). For 4-chlorobenzoate, it was catabolized via the classic hydrolytic dehalogenation pathway in strain 4-CBA. While, strain 4-CBA seemed to have a more compact gene cluster of fcbABC compared to previously reported strains. This study provides new insights into the catabolic diversity of structurally similar isomers in a contaminated niche.

Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB

Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO) genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

Crystallization and preliminary X-ray diffraction studies of the reduced form of the terminal oxygenase component of the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase.

The initial reaction of bacterial carbazole degradation is catalysed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase, ferredoxin and ferredoxin reductase components. The reduced form of the terminal oxygenase component was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals diffracted to a resolution of 1.74 Å and belonged to space group P6(5), with unit-cell parameters a = b = 92.0, c = 243.6 Å. The asymmetric unit contained a trimer of terminal oxygenase molecules.

An insight into the origin and functional evolution of bacterial aromatic ring-hydroxylating oxygenases

Bacterial aromatic ring-hydroxylating oxygenases (RHOs) are multicomponent enzyme systems which have potential utility in bioremediation of aromatic compounds in the environment. To cope with the enormous diversity of aromatic compounds in the environment, this enzyme family has evolved remarkably exhibiting broad substrate specificity. RHOs are multicomponent enzymes comprising of a homo- or hetero-multimeric terminal oxygenase and one or more electron transport (ET) protein(s). The present study attempts in depicting the evolutionary scenarios that might have occurred during the evolution of RHOs, by analyzing a set of available sequences including those obtained from complete genomes. A modified classification scheme identifying four new RHO types has been suggested on the basis of their evolutionary and functional behaviours, in relation to structural configuration of substrates and preferred oxygenation site(s). The present scheme emphasizes on the fact that the phylogenetic affiliation of RHOs is distributed among four distinct ‘Similarity classes’, independent of the constituent ET components. Similar combination of RHO components that was previously considered to be equivalent and classified together [Kweon et al., BMC Biochemistry 9, 11 (2008)] were found here in distinct similarity classes indicating the role of substrate-binding terminal oxygenase in guiding the evolution of RHOs irrespective of the nature of constituent ET components. Finally, a model for evolution of the multicomponent RHO enzyme system has been proposed, beginning from genesis of the terminal oxygenase components followed by recruitment of constituent ET components, finally evolving into various ‘extant’ RHO types.

Phylogenetic analysis reveals the surprising diversity of an oxygenase class

As metalloenzymes capable of transforming a broad range of substrates with high stereo- and regio-specificity, the multicomponent Rieske oxygenases (ROs) have been studied in bacterial systems for applications in bioremediation and industrial biocatalysis. These studies include genetic and biochemical investigations, determination of enzyme structure, phylogenetic analysis, and enzyme classification. Although RO terminal oxygenase components (RO-Os) share a conserved domain structure, their sequences are highly divergent and present significant challenges for identification and classification. Herein, we present the first global phylogenetic analysis of a broad range of RO-Os from diverse taxonomic groups. We employed objective, structure-based criteria to significantly reduce the inclusion of erroneously aligned sequences in the analysis. Our findings reveal that RO biochemical studies to date have been largely concentrated in an unexpectedly narrow portion of the RO-O sequence landscape. Additionally, our analysis demonstrates the existence two distinct groups of RO-O sequences. Finally, the sequence diversity recognized in this study necessitates a new RO-O classification scheme. We therefore propose a P450-like naming system. Our results reveal a diversity of sequence and potential catalytic functionality that has been wholly unappreciated in the RO literature. This study also demonstrates that many commonly used bioinformatic tools may not be sufficient to analyze the vast amount of data available in current databases. These findings facilitate the expanded exploration of RO catalytic capabilities in both biological and technological contexts and increase the potential for practical exploitation of their activities.

Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

Rieske nonheme monooxygenase 3-ketosteroid 9α-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshA(H37Rv) of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a β sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the β sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 β-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities.

Development of a strain for efficient degradation of polychlorinated biphenyls by patchwork assembly of degradation pathways

Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.

Cloning and Nucleotide Sequences of Carbazole Degradation Genes from Marine Bacterium Neptuniibacter sp. Strain CAR-SF

The marine bacterium Neptuniibacter sp. strain CAR-SF utilizes carbazole as its sole carbon and nitrogen sources. Two sets of clustered genes related to carbazole degradation, the upper and lower pathways, were obtained. The marine bacterium genes responsible for the upper carbazole degradation pathway, carAa, carBa, carBb, and carC, encode the terminal oxygenase component of carbazole 1,9a-dioxygenase, the small and large subunits of the meta-cleavage enzyme, and the meta-cleavage compound hydrolase, respectively. The genes involved in the lower degradation pathway encode the anthranilate dioxygenase large and small subunit AntA and AntB, anthranilate dioxygenase reductase AntC, 4-oxalocrotonate tautomerase, and catechol 2,3-dioxygenase. Reverse transcription-polymerase chain reaction confirmed the involvement of the isolated genes in carbazole degradation. Escherichia coli cells transformed with the CarAa of strain CAR-SF required ferredoxin and ferredoxin reductase for biotransformation of carbazole. Although carAc, which encodes the ferredoxin component of carbazole 1,9a-dioxygenase, was not found immediately downstream of carAaBaBbC, the carAc-like gene may be located elsewhere based on Southern hybridization. This is the first report of genes involved in carbazole degradation isolated from a marine bacterium.

Characterization of the Isophthalate Degradation Genes of Comamonas sp. Strain E6

The isophthalate (IPA) degradation gene cluster (iphACBDR) responsible for the conversion of IPA into protocatechuate (PCA) was isolated from Comamonas sp. strain E6, which utilizes phthalate isomers as sole carbon and energy sources via the PCA 4,5-cleavage pathway. Based on amino acid sequence similarity, the iphA, iphC, iphB, iphD, and iphR genes were predicted to code for an oxygenase component of IPA dioxygenase (IPADO), a periplasmic IPA binding receptor, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, a reductase component of IPADO, and an IclR-type transcriptional regulator, respectively. The iphACBDR genes constitute a single transcriptional unit, and transcription of the iph catabolic operon was induced during growth of E6 on IPA. The iphA, iphD, and iphB genes were expressed in Escherichia coli. Crude IphA and IphD converted IPA in the presence of NADPH into a product which was transformed to PCA by IphB. These results suggested that IPADO is a two-component dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) and that iphB encodes the 1,5-DCD dehydrogenase. Disruption of iphA and iphB resulted in complete loss of growth of E6 on IPA. Inactivation of iphD significantly affected growth on IPA, and the iphC mutant did not grow on IPA at neutral pH. These results indicated that the iphACBD genes are essential for the catabolism of IPA in E6. Disruption of iphR resulted in faster growth of E6 on IPA, suggesting that iphR encodes a repressor for the iph catabolic operon. Promoter analysis of the operon supported this notion.

Specific Interactions between the Ferredoxin and Terminal Oxygenase Components of a Class IIB Rieske Nonheme Iron Oxygenase, Carbazole 1,9a-Dioxygenase

Abstract Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (Oxy), ferredoxin (Fd), and ferredoxin reductase (Red) components and is a member of the Rieske nonheme iron oxygenases. Rieske nonheme iron oxygenases are divided into five subclasses (IA, IB, IIA, IIB, and III) based on the number of constituents and the nature of their redox centers. Each component of a class IIB CARDO from Nocardioides aromaticivorans IC177 was purified, and the interchangeability of the electron transfer reactions with each component from the class III CARDOs was investigated. Despite the fact that the Fds of both classes are Rieske-type, strict specificities between the Oxy and Fd components were observed. On the other hand, the Fd and Red components were interchangeable, even though the Red components differ in cofactor composition; the class IIB Red contains flavin-adenine-dinucleotide (FAD)- and NADH-binding domains, whereas the class III Red has a chloroplast-type [2Fe–2S] cluster in addition to the FAD- and NADH-binding domains. The crystal structures of the class IIB Oxy and Fd components were compared to the previously reported Fd:Oxy complex structure of class III CARDO. This comparison suggested residues in common between class IIB and class III CARDOs that are important for interactions between Fd and Oxy. In the class IIB CARDOs, these included His75 and Glu71 in Fd and Lys20 and Glu357 in Oxy for electrostatic interactions, and Phe74 and Pro90 in Fd and Trp21, Leu359, and Val367 in Oxy for hydrophobic interactions. The residues that formed the interacting surface but were not conserved between classes were thought to be necessary to form the appropriate geometry and to determine electron transfer specificity between Fd and Oxy.

Characterization of a Second Rhodococcus erythropolis SQ1 3-Ketosteroid 9α-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB

Previously we have characterized 3-ketosteroid 9alpha-hydroxylase (KSH), a key enzyme in microbial steroid degradation in Rhodococcus erythropolis strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the kshA1 gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the kshA2 gene in mutant strain RG2 restored growth on AD and ADD, indicating that kshA2 also encodes KSH activity. The functional complementation was shown to be dependent on the presence of kshB. Transcriptional analysis showed that expression of kshA2 is induced in parent strain R. erythropolis SQ1 in the presence of AD. However, promoter activity studies, using beta-lactamase of Escherichia coli as a convenient transcription reporter protein for Rhodococcus, revealed that the kshA2 promoter in fact is highly induced in the presence of 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of kshA2 in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third kshA homologue was additionally identified in a kshA1 kshA2 double gene deletion mutant strain of R. erythropolis SQ1. The developed degenerate PCR primers for kshA may be useful for isolation of kshA homologues from other (actino) bacteria.

Enzymatic Properties of Terephthalate 1,2-Dioxygenase ofComamonassp. Strain E6

The tphA1 II and tphA2 II A3 II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1 II and TphA2 II A3 II, indicating that TPADO consists of a reductase (TphA1 II) and a terminal oxygenase component (TphA2 II and TphA3 II). TphA1(II) contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1 II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe2+ was required for it. The Km values for TPA and the Vmax were determined to be 72+/-6 microM and 9.87+/-0.06 U/mg respectively.

Crystallization and preliminary X-ray diffraction studies of a novel ferredoxin involved in the dioxygenation of carbazole byNovosphingobiumsp. KA1

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 C222(1) and diffraction data were collected to a resolution of 1.9 A (the diffraction limit was 1.6 A).

Crystallization and preliminary crystallographic analysis of the ferredoxin component of carbazole 1,9a-dioxygenase fromNocardioides aromaticivoransIC177

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 A resolution and belong to space group P4(1)2(1)2.

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1

BackgroundThe 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.ResultsIn 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.ConclusionThis 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.

2P010 分子動力学シミュレーションを用いたCARDOの酸化酵素部位の解析(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

2P010 分子動力学シミュレーションを用いたCARDOの酸化酵素部位の解析(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

Crystallization and preliminary X-ray diffraction studies of the terminal oxygenase component of carbazole 1,9a-dioxygenase fromNocardioides aromaticivoransIC177

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 A resolution and belong to space group C2.

Functional and transcriptional analyses of the initial oxygenase genes for acenaphthene degradation from Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene as its sole carbon and energy source. To isolate the genes responsible for acenaphthene degradation, transposon mutagenesis was performed on strain A4 and four mini-Tn5-inserted mutants lacking the ability to utilize acenaphthene were isolated. In three of the four mini-Tn5 inserted mutants, the mini-Tn5s were inserted into the same locus (within about 16 kb) as the arhA1A2 genes, which had previously been identified as the genes encoding the terminal oxygenase components for the initial oxygenation of acenaphthene. The nucleotide sequence analysis of the corresponding 16.4 kb DNA fragment revealed the existence of 16 ORFs and a partial ORF. From these ORFs, the genes encoding the ferredoxin (ArhA3) and ferredoxin reductase (ArhA4) complementary to ArhA1A2 were identified. RT-PCR analysis suggested that a 13.5 kb gene cluster, consisting of 13 ORFs and including all the arhA genes, forms an operon, although it includes several ORFs that are apparently unnecessary for acenaphthene degradation. Furthermore, using gene disruption and quantitative RT-PCR analyses, the LysR-type activator, ArhR, required for expression of the 13.5 kb gene cluster was also identified. Transcription of the gene cluster by ArhR was induced in the presence of acenaphthene (or its metabolite), and a putative binding site (T-N11-A motif) for ArhR was found upstream from the transcription start point of arhA3.