Aromatic Ring-hydroxylating Dioxygenases Research Articles

Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

BackgroundPolycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination.ResultsGenes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus.ConclusionThese results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.

Microbial diversity in natural asphalts of the Rancho La Brea Tar Pits.

Bacteria commonly inhabit subsurface oil reservoirs, but almost nothing is known yet about microorganisms that live in naturally occurring terrestrial oil seeps and natural asphalts that are comprised of highly recalcitrant petroleum hydrocarbons. Here we report the first survey of microbial diversity in ca. 28,000-year-old samples of natural asphalts from the Rancho La Brea Tar Pits in Los Angeles, CA. Microbiological studies included analyses of 16S rRNA gene sequences and DNA encoding aromatic ring-hydroxylating dioxygenases from two tar pits differing in chemical composition. Our results revealed a wide range of phylogenetic groups within the Archaea and Bacteria domains, in which individual taxonomic clusters were comprised of sets of closely related species within novel genera and families. Fluorescent staining of asphalt-soil particles using phylogenetic probes for Archaea, Bacteria, and Pseudomonas showed coexistence of mixed microbial communities at high cell densities. Genes encoding dioxygenases included three novel clusters of enzymes. The discovery of life in the tar pits provides an avenue for further studies of the evolution of enzymes and catabolic pathways for bacteria that have been exposed to complex hydrocarbons for millennia. These bacteria also should have application for industrial microbiology and bioremediation.

Structure and Increased Thermostability ofRhodococcussp. Naphthalene 1,2-Dioxygenase

Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The structure of the Rhodococcus enzyme reveals that, despite a approximately 30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 A. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to the Rhodococcus enzyme in the same orientation as in the Pseudomonas enzyme. Circular dichroism spectroscopy experiments show that the Rhodococcus enzyme has higher thermostability than the naphthalene dioxygenase from Pseudomonas species. The Pseudomonas enzyme has an apparent melting temperature of 55 degrees C while the Rhodococcus enzyme does not completely unfold even at 95 degrees C. Both enzymes, however, show similar unfolding behavior in urea, and the Rhodococcus enzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of the Rhodococcus enzyme may be attributed to a larger buried surface area and extra salt bridge networks between the alpha and beta subunits in the Rhodococcus enzyme.

Variations in the abundance and identity of class II aromatic ring‐hydroxylating dioxygenase genes in groundwater at an aromatic hydrocarbon‐contaminated site

The abundance of genes encoding aromatic ring-hydroxylating dioxygenases (RHDs) in the groundwater at an aromatic hydrocarbon-contaminated landfill near Sydney, Australia, was determined by quantitative DNA-DNA hybridization using class II RHD genes as probes. There were marked differences in hybridization signal intensity against DNA extracted from the groundwater at seven different locations across this heterogeneous site. This was interpreted as indicating variation in RHD gene abundance. Clone libraries of polymerase chain reaction (PCR)-amplified RHD gene fragments were constructed from DNA from each of the groundwater samples. The libraries from the samples with greater RHD gene abundance were dominated by a group of bacterial class II RHD genes, designated the S-cluster, that has yet to be found in cultured isolates. These groundwater samples contained no detectable petroleum hydrocarbons. A second group of class II RHD gene sequences, designated the T-cluster, dominated RHD gene clone libraries prepared from groundwater samples that contained detectable levels of total petroleum and aromatic hydrocarbons but lower RHD gene abundance. The hosts and in situ expression of these novel genes, and the substrates of the enzymes they encode, remain unknown. The scarcity of genes from known aromatic hydrocarbon-degrading bacteria and the numerical dominance of the novel genes suggest that the hosts of these novel genes may play an important role in aromatic hydrocarbon degradation at this site.

Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp. strain A5.

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.

Detection of known and novel genes encoding aromatic ring-hydroxylating dioxygenases in soils and in aromatic hydrocarbon-degrading bacteria

Primers were designed and successfully used to screen aromatic hydrocarbon-degrading bacteria for the presence of class II aromatic ring-hydroxylating dioxygenase (RHD) genes and to amplify novel RHD genes from DNA extracted from soil using the polymerase chain reaction. Two previously undiscovered groups of genes encoding putative class II RHDs, designated the S and T clusters, were found in RHD different soil samples. Only one of 70 RHD gene fragments amplified from these soil samples could be assigned to a cluster of previously reported RHD genes. These results suggest that distinct and potentially numerically dominant groups of as-yet unrecognized aromatic hydrocarbon-degrading bacteria exist in soils.

A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9.

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 alpha and beta 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 alpha 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 alpha 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.

A Novel Suppressor of Cell Death in Plants Encoded by the Lls1 Gene of Maize

The lls1 (lethal leaf spot1) locus of maize is defined by a recessive mutation characterized by the initiation, in a developmentally programmed manner, of necrotic lesions that expand to kill leaves cell autonomously. The loss-of-function nature of all lls1 mutants implies that the Lls1 gene is required to limit the spread of cell death in mature leaves. We have cloned the Lls1 gene by tagging with Mutator, a transposable element system in maize, and we show that it encodes a novel protein highly conserved in plants. Two consensus binding motifs of aromatic ring-hydroxylating dioxygenases are present in the predicted LLS1 protein, suggesting that it may function to degrade a phenolic mediator of cell death.

Structure-function Analysis of the Bacterial Aromatic Ring-hydroxylating Dioxygenases

The bacterial aromatic ring-hydroxylating dioxygenases represent a broad range of multi-component enzymes that possess many properties and mechanisms in common. This has enabled their classification based on structural properties. This classification has been supported by sequence comparisons based on nucleotide and primary amino-acid sequences. Comparison of the low homology sequences has also enabled the identification of highly conserved functional motifs—for example iron-sulphur ligation domains—whereas analysis of high homology sequences has allowed the characterization of amino acids involved in more specific functions, such as the control of substrate specificity. This combination of sequence analysis with structure-function studies proves to be invaluable for future protein engineering of improved enzymes for both biocatalysis and environmental remediation. The chapter focuses on the ring-hydroxylating dioxygenases, referring to ring-cleavage dioxygenases and other related systems, and also describes bacterial oxygenases and dioxygenases.

Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).

The evolutionary relationship of biphenyl dioxygenase from Gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from Gram-negative bacteria

The Gram + bacterium Rhodococcus globerulus P6 (RgP6) catabolizes a range of polychlorinated biphenyl (PCB) congeners, thus being of interest in bioelimination processes for PCB. The first step in the pathway, a dioxygenase attack of one of the biphenyl (BP) rings, is catalyzed by biphenyl dioxygenase (BDO). In this study, the nucleotide (nt) sequences of the four clustered cistrons, bphA1 A2A3A4, encoding the subunits of BDO and forming part of the bph operon of RgP6 for BP degradation, were determined. A conserved motif proposed to bind a Rieske-type [2Fe-2s]cluster was identified in the deduced amino acid (aa) sequence of both the a subunit of the terminal oxygenase (BphA1) and ferredoxin (BphA3). The ferredoxin reductase subunit (BphA4) contains conserved sites for FAD and NADH binding. Deduced as sequences of the BDO subunits shared homologies to multicomponent aromatic ring-hydroxylating dioxygenases from Gram − microorganisms. Stronger identity was found to toluene dioxygenase (TDO) of Pseudomonas putida F1 than to other BDO. Aa sequence comparisons suggest that BP degradation genes of RgP6 may have originated in Gram − microorganisms, probably Pseudomonas, and subsequently transferred to this Gram + bacterium.