Biphenyl Dioxygenase Research Articles

Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.

Oxidation of 2-methoxynaphthalene by toluene, naphthalene and biphenyl dioxygenases: structure and absolute stereochemistry of metabolites

2-Methoxynaphthalene was subjected to biooxidation by whole cells of six organisms: Pseudomonas putida F39/D containing toluene dioxygenase, Escherichia coli JM109(pDTG601), containing recombinant toluene dioxygenase from Pp F39/D, Pseudomonas sp. NCIB 9816/11, containing naphthalene dioxygenase, E. coli JM109(pDTG141), containing recombinant naphthalene dioxygenase from NCIB 9816/11, E. coli C534(ProR/Sac) containing recombinant naphthalene dioxygenase from Pp G7, and Beijerinckia sp. B8/36, containing biphenyl dioxygenase. The major product of oxidation by the naphthalene and biphenyl dioxygenases has been isolated and identified as (1 R,2 S)-dihydroxy-7-methoxy-1,2-dihydronaphthalene, 2c. A minor product, (1 R,2 S)-dihydroxy-6-methoxy-1,2-dihydronaphthalene, 3c, has also been detected. Oxidation by the toluene dioxygenase-containing organisms led to the isolation of 3c as the major product. Minor products detected in these reactions were 2c, and a third compound, (1 S,2 S)-dihydroxy-3-methoxy-1,2-dihydronaphthalene, 4c. Structural studies and dehydration of the diols to a mixture of naphthols are described. The absolute stereochemistry of these new diols has been established by correlation with known compounds. The organisms' potential in the production of new metabolites as useful chiral synthons by biooxidation of 2-substituted naphthalenes is indicated.

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).

Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp. strain KKS102 was determined. Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems. When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected. The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity. These data clearly suggest that ORF2 encodes ferredoxin reductase. The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown.

Construction of hybrid biphenyl ( bph) and toluene ( tod) genes for functional analysis of aromatic ring dioxygenases

Multicomponent enzyme complexes of biphenyl (BP) dioxygenase (Dox) encoded by the gene cluster, bphA1 A2A3A4 in Pseudomonas pseudoalcaligenes strain KF707 [Taira et al., J. Biol. Chem. 267 (1992) 4844–4858] and toluene Dox encoded by the gene cluster, todC1C2BA in P. putida strain F1 [Zylstra et al., J. Biol. Chem. 264 (1989) 14940–14946], show high homologies (approx. 60%) for the corresponding subunit component in spite of the fact that they have discrete substrate specificities. We constructed hybrid gene clusters by replacing the gene component (s) between the large and small subunits of terminal Dox in the bph and tod gene clusters, and analyzed the function of a novel hybrid aromatic ring Dox. Escherichia coli cells expressing the hybrid gene clusters, todC1::bphA2A3A4, todC1C2::bphA3A4 and bphA1::todC2::bphA3A4, gained the ability to convert benzene-toluene and their derivatives to the dihydrodiols, indicating that the hybrid terminal Dox composed of TodCI::BphA2 and BphA1::TodC2 forms a functionally active multicomponent Dox associated with ferredoxin (Fer) (BphA3) and Fer reductase (BphA4). Moreover, hybrid Dox (composed of TodCl::BphA2A3A4 and TodC1C2::BphA3A4) showed a wide substrate specificity rather similar to that of the wild-type toluene Dox (TodClC2BA). On the other hand, the hybrid Dox (BphA1::TodC2::BphA3A4) showed oxidative activities for the same compounds, but the rate of oxidation was dependent upon the substrate. These results suggest that ( i) the two subunits of terminal Dox are critically involved in the substrate specificity for BP, benzene and their derivatives, and ( ii) the electron transport proteins, Fer and Fer reductase, are exchangeable with one another between the BP Dox and toluene Dox complexes.

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

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

Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene.

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.

Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation.

The cistronic organization of the bph locus, encoding a biphenyl/polychlorinated biphenyl (PCB) degradation pathway in Pseudomonas sp. LB400, has been elucidated. Seven structural genes, encoding biphenyl dioxygenase (bphA1A2A3A4), biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (bphB), biphenyl-2,3-diol-1,2-dioxygenase (bphC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD), have been located. The complete sequences of bphB, bphC and bphD are reported. Taken together with the data of Erickson and Mondello [J. Bacteriol. 174 (1992) 2903-2912], Pseudomonas sp. LB400 is now the first strain for which the sequences of all genes encoding the catabolism from biphenyls to benzoates have been determined. Comparisons of the deduced amino acid (aa) sequences of BphB, BphC and BphD with those of related proteins led to predictions about catalytically important aa residues. Six Bph have been detected and identified. Five of them could be obtained as the most abundant proteins when their genes were expressed in Escherichia coli.

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.

Use of a genetically engineered Escherichia coli strain to produce 1,2-dihydroxy-4'-chlorobiphenyl

Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.

Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707.

The entire nucleotide sequences (6.8 kilobase pairs) of the bphABC genes and their products involved in the initial dioxygenation and ring-meta-cleavage of biphenyls and polychlorinated biphenyls were determined. The first bphA gene starts about a 100 base pairs downstream from the transcriptional initiation site. The bphA region, which encodes a cluster of enzymes including biphenyl dioxygenase catalyzing the initial catabolic step, consists of five open reading frames (ORFs). Five proteins corresponding to these ORFs in the molecular masses were detected by in vitro protein synthesis, of which four ORFs are very similar to the recently reported todC1C2BA genes coding for the corresponding enzymes catalyzing the initial dioxygenation reactions of toluene (Zylstra, G.J., and Gibson, D. T. (1989) J. Biol. Chem. 264, 14940-14946). The third open reading frame (ORF3) of the bphA region, missing its counterpart in the toluene dioxygenase gene cluster, was site-specifically deleted, and the resulting enzymatically active mutant reveals that this ORF3 is not mandatory for the catabolism of biphenyls. Thus the biphenyl degradation pathway and the responsible enzymes/genes are very similar to those of toluene degradation despite their discrete substrate specificity.

Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida

Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds

A bioconversion process was demonstrated by manipulation of catabolic genes. Catabolic intermediates of various biphenyl compounds could be efficiently produced by Pseudomonas aeruginosa carrying recombinant plasmids containing a set of cloned bph genes. A dihydrodiol compound was produced by the strain carrying plasmid pMFB4 containing bphA (encoding biphenyl dioxygenase) gene. A dihydroxy compound was produced from 4-chlorobiphenyl by the strain carrying plasmid pMFB6 containing bphA and bphB (encoding dihydrodiol dehydrogenase) genes. Tetrahydroxybiphenyl was accumulated as the final product via dihydroxybiphenyl from biphenyl by the same pMFB6 carrying strain. Meta-cleavage yellow compounds were produced from biphenyl and its derivatives substituted with methyl, chloro, bromo, or nitro group on one of the biphenyl rings by the strain carrying plasmid pMFB2 containing bphA, bphB and bphC (encoding dihydroxybiphenyl dioxygenase) genes.

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.