Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase genes.

The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase (EC 1.13.11.3) were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5-kilobase-pair PstI fragment into the broad-host-range cloning vector pRO2317. The resultant clone was able to complement protocatechuate 3,4-dioxugenase mutations in P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host. Since the cloned genes exhibited normal induction patterns when present in P. cepacia DBO1, it was concluded that induction was subject to negative control. Regulatory studies with P. cepacia wild-type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate. Further studies of P. cepacia DBO1 showed that p-hydroxybenzoate hydroxylase (EC 1.14.13.2), the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hydroxybenzoate and beta-ketoadipate.     

Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.     

Enhanced detection and characterization of protocatechuate 3,4-dioxygenase in Acinetobacter lwoffii K24 by proteomics using a column separation

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.     

Cloning of the genes for a 4-sulphocatechol-oxidizing protocatechuate 3,4-dioxygenase from Hydrogenophaga intermedia S1 and identification of the amino acid residues responsible for the ability to convert 4-sulphocatechol

The genes for a protocatechuate 3,4-dioxygenase (P34O-II) with the ability to oxidize 4-sulphocatechol were cloned from the 4-aminobenzenesulphonate(sulphanilate)-degrading bacterium Hydrogenophaga intermedia strain S1 (DSMZ 5680). Sequence comparisons of the deduced amino acid sequences of both subunits of the P34O-II from H. intermedia S1 (PcaH-II and PcaG-II) with those of another P34O-II, previously obtained from Agrobacterium radiobacter S2, and the corresponding sequences from the protocatechuate 3,4-dioxygenases from other bacterial genera demonstrated that seven amino acid residues, which were conserved in all previously known P34Os (P34O-Is), were different in both P34O-IIs. According to previously published structural data for the P34O of Pseudomonas putida only two of these amino acid residues were located near the catalytical centre. The respective amino acid residues were mutated in the P34O-I from A. radiobacter S2 by site-specific mutagenesis, and it was found that a single amino acid exchange enabled the protocatechuate converting P34O also to oxidize 4-sulphocatechol.     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC).

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199

Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.     

Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes.

The genes that encode the alpha and beta subunits of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.3]) were cloned from a Pseudomonas putida (formerly P. aeruginosa) (ATCC 23975) genomic library prepared in lambda phage. Plaques were screened by hybridization with degenerate oligonucleotides designed using known amino acid sequences. A 1.5-kb SmaI fragment from a 15-kb primary clone was subcloned, sequenced, and shown to contain two successive open reading frames, designated pcaH and pcaG, corresponding to the beta and alpha subunits, respectively, of 3,4-PCD. The amino acid sequences deduced from pcaHG matched the chemically determined sequence of 3,4-PCD in all except three positions. Cloning of pcaHG into broad-host-range expression vector pKMY319 allowed high levels of expression in P. putida strains, as well as in Proteus mirabilis after specific induction of the plasmid-encoded nahG promoter with salicylate. The recombinant enzyme was purified and crystallized from P. mirabilis, which lacks an endogenous 3,4-PCD. The physical, spectroscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, the same transient enzyme intermediates were formed during the catalytic cycle. These studies establish the methodology which will allow mechanistic investigations to be pursued through site-directed mutagenesis of P. putida 3,4-PCD, the only aromatic ring-cleaving dioxygenase for which the three-dimensional structure is known.     

Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS.

The two-component nonheme iron dioxygenase system 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS catalyzes the double hydroxylation of 2-halobenzoates with concomitant release of halogenide and carbon dioxide, yielding catechol. The gene cluster encoding this enzyme, cbdABC, was localized on a 70-kbp conjugative plasmid designated pBAH1. The nucleotide sequences of cbdABC and flanking regions were determined. In the deduced amino acid sequence of the large subunit of the terminal oxygenase component (CbdA), a conserved motif proposed to bind the Rieske-type [2Fe-2S] cluster was identified. In the NADH:acceptor reductase component (CbdC), a putative binding site for a chloroplast-type [2Fe-2S] center and possible flavin adenine dinucleotide- and NAD-binding domains were identified. The cbdABC sequences show significant homology to benABC, which encode benzoate 1,2-dioxygenase from Acinetobacter calcoaceticus (52% identity at the deduced amino acid level), and to xylXYZ, which encode toluate 1,2-dioxygenase from Pseudomonas putida mt-2 (51% amino acid identity). Recombinant pkT231 harboring cbdABC and flanking regions complemented a plasmid-free mutant of wild-type P. cepacia 2CBS for growth on 2-chlorobenzoate, and it also allowed recombinant P. putida KT2440 to metabolize 2-chlorobenzoate. Functional NADH:acceptor reductase and oxygenase components of 2-halobenzoate 1,2-dioxygenase were enriched from recombinant Pseudomonas clones.     

Peptide mass fingerprinting- and 2-DE/MS-based analysis of the biodegradation potential for monocyclic aromatic hydrocarbons in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

The combined analysis of peptide mass fingerprinting and 2-DE/MS using the induced and selected protein spots following growth of Pseudomonas sp. DU102 on benzoate or p-hydroxybenzoate revealed not only alpha- and beta-subunits of protocatechuate 3,4-dioxygenase but also catechol 1,2-dioxygenase responsible for ortho-pathway through ring-cleavage of aromatic compounds. Toluate 1,2-dioxygenase and p-hydroxybenzoate hydroxylase were also identified. Purification of intradiol dioxygenases such as catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase from the benzoate or p-hydroxybenzoate culture makes it possible to trace the biodegradation pathway of strain DU102 for monocyclic aromatic hydrocarbons. Interestingly, vanillin-induced protocatechuate 3,4-dioxygenase was identical in amino acid sequences with protocatechuate 3,4-dioxygenase from p-hydroxybenzoate.     

DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA: evidence for evolutionary divergence of intradiol dioxygenases by acquisition of DNA sequence repetitions.

The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.     

Phenol degradation by halophilic bacteria isolated from hypersaline environments

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.     

Catechol 1,2-dioxygenase from the new aromatic compounds - Degrading Pseudomonas putida strain N6

This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K_m and V_max was 85.19 ??M and 14.54 ??M min⁻¹ respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.     

Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases.

The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.     

Burkholderia sp. NCIMB 10467菌株中原儿茶酸3, 4-双加氧酶基因的分子克隆和生化特性研究（英文稿）

NCIMB 10467是一株木质素降解菌, 根据其16S rDNA序列将其重新分类为Burkholderia菌属。研究显示, 在NCIMB 10467菌株中, 不同的底物可以诱导该菌株对于原儿茶酸的多种代谢形式。根据克隆到的一段原儿茶酸邻位开环酶, 即原儿茶酸3, 4-双加氧酶(P34D; EC 1.13.11.3) a-亚基的保守序列, 通过染色体步移的方法, 得到一段9505 bp的DNA片段。序列分析显示, 在这段9.5 kb的DNA片段中, 两个可能的开放阅读框pcaG 和 pcaH分别编码P34D的a-亚基和b-亚基。将pcaGH克隆并在大肠杆菌中进行表达后, 可以检测到P34D的活性。而pcaH在NCIMB 10467菌株中的敲除则使该菌完全丧失了代谢原儿茶酸的能力。由此证实, 克隆到的pcaGH基因确实编码原儿茶酸3, 4-双加氧酶, 并且对于NCIMB 10467菌株对原儿茶酸的代谢是必需的。     

Burkholderia sp. NCIMB 10467菌株中原儿茶酸3, 4-双加氧酶基因的分子克隆和生化特性研究

NCIMB 10467是一株木质素降解菌, 根据其16S rDNA序列将其重新分类为Burkholderia菌属。研究显示, 在NCIMB 10467菌株中, 不同的底物可以诱导该菌株对于原儿茶酸的多种代谢形式。根据克隆到的一段原儿茶酸邻位开环酶, 即原儿茶酸3, 4-双加氧酶(P34D; EC 1.13.11.3) a-亚基的保守序列, 通过染色体步移的方法, 得到一段9505 bp的DNA片段。序列分析显示, 在这段9.5 kb的DNA片段中, 两个可能的开放阅读框pcaG 和 pcaH分别编码P34D的a-亚基和b-亚基。将pcaGH克隆并在大肠杆菌中进行表达后, 可以检测到P34D的活性。而pcaH在NCIMB 10467菌株中的敲除则使该菌完全丧失了代谢原儿茶酸的能力。由此证实, 克隆到的pcaGH基因确实编码原儿茶酸3, 4-双加氧酶, 并且对于NCIMB 10467菌株对原儿茶酸的代谢是必需的。     

Characterization of protocatechuate 4,5-dioxygenase induced from p-hydroxybenzoate-cultured Pseudomonas sp. K82

Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, p-hydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 (alpha subunit and beta subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer (alpha1beta1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 degrees C. PCR amplification of these two subunits of PCD4,5 revealed that the alpha subunit and beta subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.     

The complete amino acid sequence of the beta-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

The complete amono aicd sequence of the beta-subunit of protocatechuate 3,4-dioxygenase is presented. The beta-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated beta-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphyloccal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated beta-subunit. The calculated molecular weight was 26,588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Purification and gene cloning of the oxygenase component of the terephthalate 1,2-dioxygenase system from Delftia tsuruhatensis strain T7

The terephthalate 1,2-dioxygenase system (TERDOS) was found in cell extracts of Delftia tsuruhatensis strain T7 (=IFO16741) grown in terephthalate-salt medium. The cell extract was separated by anion exchange chromatography to yield two fractions (R and Z) that were necessary for oxygenation of terephthalate with NADH and Fe(2+). The oxygenase component of TERDOS (TerZ) was purified from fraction Z by gel filtration chromatography to near homogeneity. An alpha(3)beta(3) subunit structure was deduced from the molecular masses of 235, 46 and 17 kDa of the native complex and the alpha- and beta-subunits, respectively. The N-terminal amino acid sequences of the two subunits of TerZ allowed polymerase chain reaction primers to be deduced and the DNA sequence of the alpha-subunit was determined. The amino acid sequence of the alpha-subunit (TerZalpha) showed significant similarities to the large subunits of multicomponent ring-hydroxylating oxygenases. Two motifs in the deduced amino acid sequence, a Rieske [2Fe-2S] center and a mononuclear Fe(II) binding site, were observed. Phylogenetic analyses indicated that TerZalpha and the large oxygenase component subunits ortho-halobenzoate 1,2-dioxygenase and salicylate-5-hydroxylase form a cluster that is distant from the rest of the large oxygenase subunits of multicomponent ring-hydroxylating oxygenases.