Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Two Types of Bacteria Having Angular Dioxygenases with Different Features

Two kinds of bacteria having different-structured angular dioxygenases—a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10—were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.     

Isolation and characterization of the gene encoding the chloroplast-type ferredoxin component of carbazole 1,9a-dioxygenase from a putative Kordiimonas sp.

Carbazole (CAR)-degrading genes (carRAaCBaBb) were isolated from marine CAR-degrading isolate strain OC9 (probably Kordiimonas gwangyangensis) using shotgun cloning experiments and showed 35–65% similarity with previously reported CAR-degrading genes. In addition, a ferredoxin-like gene (carAc) was found downstream of carR, although it was not homologous with any reported ferredoxin components of the CAR 1,9a-dioxygenase (CARDO) system. The carAc-deduced amino acid sequence possessed consensus sequences for chloroplast-type iron-sulfur proteins for binding the [2Fe-2S] cluster. These car genes were arranged in the order of carAcRAaCBaBb, but carRAc and carAaCBaBb genes were the opposite orientation. Escherichia coli JM109 cells harboring pBOC91 (carAa) converted CAR to 2′-aminobiphenyl-2,3-diol at a ratio of 12%, and the transformation ratio of CAR increased from 12 to 100% when carAc was added, indicating that CarAc is the ferredoxin component of the CARDO system in strain OC9. This is the first finding of a chloroplast-type ferredoxin component in a CARDO system. Biotransformation tests with aromatic compounds revealed that the strain OC9 CarAaAc showed activity with polycyclic aromatic hydrocarbons and dioxin compounds and exhibited significant activity for fluorene, unlike previously reported CARDOs.     

Cloning and Expression of the Benzoate Dioxygenase Genes from Rhodococcus sp. Strain 19070

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.     

Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to utilize toluene, m-xylene, p-xylene, biphenyl, naphthalene, phenanthrene, and anthracene as sole sources of carbon and energy for growth. A forty kilobase region of DNA containing most of the genes for the degradation of these aromatic compounds was previously cloned and sequenced. Insertional inactivation of bphC results in the inability of B1 to grow on both polycyclic and monocyclic compounds. Complementation experiments indicate that the metabolic block is actually due to a polar effect on the expression of bphA3, coding for a ferredoxin component of a dioxygenase. Lack of the ferredoxin results in a nonfunctional polycyclic aromatic hydrocarbon dioxygenase and a nonfunctional toluate dioxygenase indicating that the electron transfer components are capable of interacting with multiple oxygenase components. Insertional inactivation of a gene for a dioxygenase oxygenase component downstream of bphA3 had no apparent effect on growth besides a polar effect on nahD which is only needed for growth of B1 on naphthalene. Insertional inactivation of either xylE or xylG in the meta-cleavage operon results in a polar effect on bphB, the last gene in the operon. However, insertional inactivation of xylX at the beginning of this cluster of genes does not result in a polar effect suggesting that the genes for the meta-cleavage pathway, although colinear, are organized in at least two operons. These experiments confirm the biological role of several genes involved in metabolism of aromatic compounds by S. yanoikuyae B1 and demonstrate the interdependency of the metabolic pathways for polycyclic and monocyclic aromatic hydrocarbon degradation. Received 13 May 1999/ Accepted in revised form 05 July 1999     

Detailed comparison between the substrate specificities of two angular dioxygenases, dibenzofuran 4,4a-dioxygenase from Terrabacter sp. and carbazole 1,9a-dioxygenase from Pseudomonas resinovorans

The preferred substrates in angular dioxygenation, monooxygenation, and lateral dioxygenation by dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas resinovorans strain CA10 are shown to be distinctly different. The preferred oxygenation reactions suggest that DFDO evolved from a polycyclic aromatic hydrocarbon dioxygenase and that its most preferred substrates were fluorene and 9-fluorenone. The angular dioxygenases involved in the degradation pathway of dibenzofuran (dioxin) and fluorene are closely related in function, while CARDO is a novel enzyme not only phylogenetically but also functionally.     

Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10.

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.     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

Summary The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.     

Electron transfer complex formation between oxygenase and ferredoxin components in Rieske nonheme iron oxygenase system

Carbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonheme iron oxygenase system (ROS), consists of a terminal oxygenase (CARDO-O) and electron transfer components (ferredoxin [CARDO-F] and ferredoxin reductase [CARDO-R]). We determined the crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of CARDO-O with its electron donor, CARDO-F, at 1.9, 1.8, and 2.0 A resolutions, respectively. These structures provide the first structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in ROS. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole resulted in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site.     

Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

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 phnA1_a 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.     

Characterization of the meta-Cleavage Compound Hydrolase Gene Involved in Degradation of the Lignin-Related Biphenyl Structure by Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 has the ability to transform a lignin-related biphenyl compound, 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), to 5-carboxyvanillic acid (5CVA) via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). In the 4.9-kb HindIII fragment containing the OH-DDVA meta-cleavage dioxygenase gene (ligZ), we found a novel hydrolase gene (ligY) responsible for the conversion of the meta-cleavage compound of OH-DDVA to 5CVA. Incorporation of ¹⁸O from H₂¹⁸O into 5CVA indicated there was a hydrolytic conversion of the OH-DDVA meta-cleavage compound to 5CVA. LigY exhibited hydrolase activity only toward the meta-cleavage compound of OH-DDVA, suggesting its restricted substrate specificity.     

Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1_a and akbA2_a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1_b and akbA2_b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1_a-AkbA2_a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1_a-AkbA2_a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.     

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

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.