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.     

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Environmental polycyclic aromatic hydrocarbon (PAH) exposure and DNA damage in Mexican children

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants presenting a public health risk, particularly to children, a vulnerable population. PAHs have genotoxic and carcinogenic properties, which depend on their metabolism. Many enzymes involved in PAH metabolism, including CYP1A1, CYP1B1, GSTM and GSTT are polymorphic, which may modulate the activation/deactivation of these compounds. We evaluated PAH exposure and DNA damage in children living in the vicinity of the main petrochemical complex located in the Gulf of Mexico, and explored the modulation by genetic polymorphisms of PAH excretion and related DNA damage. The participants (n=82) were children aged 6-10y attending schools near the industrial area. Urinary 1-hydroxypyrene (1-OHP; a biomarker of PAH exposure) was determined by reverse-phase-HPLC; DNA damage by the comet assay (Olive Tail Moment (OTM) parameter); CYP1A1*2C and CYP1B1*3 polymorphisms by real time-PCR; and GSTM1*0 and GSTT1*0 by multiplex PCR. The median value of 1-OHP was 0.37μmol/mol creatinine; 59% of children had higher 1-OHP concentrations than those reported in environmentally exposed adults (0.24μmol/mol creatinine). A stratified analysis showed increased DNA damage in children with 1-OHP concentrations greater than the median value. We observed higher 1-OHP concentrations in children with CYP1A1*2C or GSTM1*0 polymorphisms, and a positive influence of CYP1A1*2C on OTM values in children with the highest PAH exposure. The data indicate that children living in the surroundings of petrochemical industrial areas are exposed to high PAH levels, contributing to DNA damage and suggesting an increased health risk; furthermore, data suggest that polymorphisms affecting activation enzymes may modulate PAH metabolism and toxicity.     

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 phnA1a 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.     

Genetics of polycyclic aromatic hydrocarbon metabolism in diverse aerobic bacteria

Polycyclic aromatic hydrocarbons (PAHs), which consist of two or more fused aromatic rings, are widespread in the environment and persist over long periods of time. The decontamination of a PAH-polluted environment is of importance because some PAHs are toxic, mutagenic, and carcinogenic and therefore are health hazards. As part of the efforts to establish remediation processes, the use of aerobic bacteria has been extensively studied, and both enzymologic and genetic studies are underway for the purpose of effective biodegradation. In the last two decades, one highly conserved group of PAH-catabolic genes from Pseudomonas species, called the nah-like genes, has been well investigated, and much has been found, including the structure-function relationships and the evolutionary trails of the catabolic enzymes. However, recently, PAH-catabolic genes, which are evolutionarily different from the nah-like genes, have been characterized from both Gram-negative bacteria other than Pseudomonas species and Gram-positive bacteria, and the information about these genes is expanding. This review is an outline of genetic knowledge about bacterial PAH catabolism.     

Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading betaproteobacterium

A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.     

Two polycyclic aromatic hydrocarbon o-quinone reductases from a pyrene-degrading Mycobacterium

Polycyclic aromatic hydrocarbon (PAH) o-quinone reductase (PQR) plays a crucial role in the detoxification of PAH o-quinones by reducing them to catechols. Two constitutive PQRs were found in cell extracts of a pyrene-degrading Mycobacterium sp. strain PYR100. The enzymes had an activity towards 9,10-phenanthrenequinone (PQ) and/or 4,5-pyrenequinone (PyQ), and the relative amounts varied with the pH of the culture media. PQR1, containing an FAD cofactor, was a monomer (20.1 kDa), and PQR2, with no flavin cofactor, was a homodimer (26.5 kDa subunits). There was no homology between the N-terminal sequences of PQR1 and PQR2. Dicumarol and quercetin inhibited PQR2 more strongly than PQR1. PQR1 had much lower specificity constants (k(cat)/K(m), 10(5)M(-1)s(-1)) for menadione (0.80) and PQ (5.19) than PQR2 (13.9 for menadione and 176 for PQ). Additionally, PQR2 exhibited a broad substrate specificity with high specificity constants for 1,4-naphthalenequinone, 1,2-naphthalenequinone, and PyQ.     

A CASE-SAR analysis of polycyclic aromatic hydrocarbon carcinogenicity

A CASE SAR analysis was performed on a selected database of PAHs to investigate the possible use of the CASE method as an aid for preliminary assessment of carcinogenic potential of untested environmental PAHs. A data set, denoted LEARN, consisting of 78 PAHs and their experimental carcinogenicities was used to 'train' the CASE method and derive the CASE fragments. 8 activating fragments and 4 inactivating fragments were identified. These fragments predicted the activities of 94% of the LEARN set correctly. The biological significance of several of these fragments are rationalized in light of the current theories of PAH carcinogenesis. Using these fragments, the potential activities of a database of 106, mostly untested PAHs, denoted TEST, were predicted. These were compared to 'expert judgement' predictions based on mechanistic considerations in order to evaluate the extent of concordance between these two methods and their respective strengths and weaknesses. Initial poor agreement (64%) was attributed to limitations of the LEARN database involving inadequate representation of 2- and 3-ring PAH subclasses. When these subclasses were excluded from the TEST database, the concordance improved to 90%. The CASE fragments were also used to predict the activities of a database of 24 PAHs, denoted VALIDATE (not included in the LEARN set) for which carcinogenicity data were available. The total prediction accuracy of 75% (89% of the actives correctly identified), despite the structural diversity of the VALIDATE set, provided independent evidence of the utility of the present CASE results. A close examination of the CASE incorrect predictions was conducted to delineate inadequancies of these CASE results in order to provide cautionary guidance for future application of the method. Finally, the present results were compared to the results of a previous CASE analysis based on a more limited PAH data set, and were found to be of greater general utility. It is concluded that the CASE fragments derived in the current study should provide a useful tool for assisting and complementing 'expert judgement' in the preliminary screening of PAHs for carcinogenic activity.     

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

The polycyclic aromatic hydrocarbon phenanthrene causes oxidative stress and alters polyamine metabolism in the aquatic liverwort Riccia fluitans L

The polycyclic aromatic hydrocarbon (PAH) phenanthrene (PHEN) is a highly toxic pollutant, commonly found in aquatic environments, the effects of which on aquatic plants have not been studied in depth. As PAHs are known to induce oxidative stress and recent studies have shown that polyamines (PAs) participate in the defence reactions protecting plants against environmental stresses, PA metabolism and oxidative damage were investigated in the aquatic form of the liverwort Riccia fluitans L. exposed to PHEN. Exposure of Riccia fluitans plants to PHEN at concentrations of 0.5 microm or less induced oxidative stress, but at a level from which plants could recover. Despite increased levels of enzymatic and non-enzymatic antioxidants, recovery appeared, at least in part, due to increased synthesis of PAs, achieved via increased activities of the enzymes arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC). Chemical inhibition of these enzymes inhibited plant recovery, while treatment with PAs aided recovery. Finally, as chloroplasts and the plasma membrane appeared to be key targets for PHEN-induced damage, the potential roles of PAs in protecting these cellular components were considered. How PAs could protect plant cells from serious environmental pollutants such as PHEN and could prevent oxidative stress is discussed.     

The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis

Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic analysis of polycyclic aromatic hydrocarbon degradation in <Emphasis Type="Italic">Mycobacterium vanbaalenii</Emphasis> PYR-1

Mycobacterium vanbaalenii PYR-1 is well known for its ability to degrade a wide range of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs). The genome of this bacterium has recently been sequenced, allowing us to gain insights into the molecular basis for the degradation of PAHs. The 6.5 Mb genome of PYR-1 contains 194 chromosomally encoded genes likely associated with degradation of aromatic compounds. The most distinctive feature of the genome is the presence of a 150 kb major catabolic region at positions 494 ~ 643 kb (region A), with an additional 31 kb region at positions 4,711 ~ 4,741 kb (region B), which is predicted to encode most enzymes for the degradation of PAHs. Region A has an atypical mosaic structure made of several gene clusters in which the genes for PAH degradation are complexly arranged and scattered around the clusters. Significant differences in the gene structure and organization as compared to other well-known aromatic hydrocarbon degraders including Pseudomonas and Burkholderia were revealed. Many identified genes were enriched with multiple paralogs showing a remarkable range of diversity, which could contribute to the wide variety of PAHs degraded by M. vanbaalenii PYR-1. The PYR-1 genome also revealed the presence of 28 genes involved in the TCA cycle. Based on the results, we proposed a pathway in which HMW PAHs are degraded into the β-ketoadipate pathway through protocatechuate and then mineralized to CO₂ via TCA cycle. We also identified 67 and 23 genes involved in PAH degradation and TCA cycle pathways, respectively, to be expressed as proteins.     

Mechanism of action of food-associated polycyclic aromatic hydrocarbon carcinogens

The polycyclic aromatic hydrocarbon carcinogens are formed in the inefficient combustion of organic matter and contaminate foods through direct deposition from the atmosphere or during cooking or smoking of foods. These potent carcinogens and mutagens require metabolism to dihydrodiol epoxide metabolites in order to express their biological activities. In vitro studies show that these reactive metabolites can react with the bases in DNA with different specificities depending upon the hydrocarbon from which they are derived. Thus, the more potent carcinogens react more extensively with adenine residues in DNA than do the less potent carcinogens, with the result that mutation at A . T base pairs is enhanced for the more potent carcinogens. In the past few years, considerable clarification of the mechanism of metabolic activation have been achieved and the focus for the immediate future is expected to be on how the reactive metabolites actually bring about biological responses.     

p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon.

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.     

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.     

Detection of polycyclic aromatic hydrocarbon degradation genes in different soil bacteria by polymerase chain reaction and DNA hybridization

Twenty different strains of Pseudomonas, Mycobacterium, Gordona, Sphingomonas, Rhodococcus and Xanthomonas which degrade polycyclic aromatic hydrocarbons (PAH) were characterized in respect to genes encoding degradation enzymes for PAH. Genomic DNA from these strains was hybridized with a fragment of ndoB, coding for the large iron sulfur protein (ISP alpha) of the naphthalene dioxygenase from Pseudomonas putida PaW736 (NCIB 9816). A group of seven naphthalene-degrading Pseudomonas strains showed strong hybridization with the ndoB probe, and five Gordona, Mycobacterium, Rhodococcus and Pseudomonas strains able to degrade higher molecular weight PAH showed weaker hybridization signals. Using a polymerase chain reaction (PCR) approach, seven naphthalene-degrading Pseudomonas strains showed a PCR fragment of the expected size with ndoB-specific primers and additionally ten strains of Gordona, Mycobacterium, Pseudomonas, Sphingomonas and Xanthomonas able to degrade higher molecular weight PAH were detected with degenerate primer-pools specific for the ISP alpha [2Fe-2S]-Rieske center of diverse aromatic hydrocarbon dioxygenases. This suggests a molecular relationship between genes coding for PAH catabolism in various PAH-degrading bacterial taxa, which could be used to evaluate the PAH-degradation potential of mixed populations.     

The discrepancy between in vivo and in vitro experiments with polycyclic aromatic hydrocarbon(PAH) carcinogens: a hypothetical explanation

The hypothesis suggested in this paper is an attempt to explain a discrepancy between in vivo and in vitro polycyclic aromatic hydrocarbons (PAH)-carcinogenesis experiments, described frequently in the literature. Whereas, in pretreated animals, a higher level of induced PAH-metabolizing enzymes reduces PAH-carcinogenicity, in the tissues or homogenates from pretreated animals the induced PAH-metabolizing enzymes increase the carcinogenic effects of PAH. In our model, both the pretreatment or route of administration should cause a frameshift in the alternation of active and inactive metabolites in the compartments. In pretreated animals the carcinogenicity of PAH administered per os is reduced, because the PAH metabolism is completed to tetrol, the ultimate inactive metabolite, before it reaches the target tissue. The hypothesis explains the discrepancy and makes predictions which can be tested experimentally.     

Polycyclic aromatic hydrocarbon oxidation during prostaglandin biosynthesis

Polycyclic hydrocarbons and other xenobiotics are cooxygenated during the oxidative metabolism of polyunsaturated fatty acids. The products of polycyclic hydrocarbon cooxygenation depend upon the hydrocarbon but they appear to be formed by radical (or electron transfer) mechanisms. The cooxygenations are hydroperoxide-dependent oxidations catalyzed by peroxidases and may be triggered by enzymes which biosynthesize fatty acid hydroperoxides. Prostaglandin endoperoxide synthetase is the principal hydroperoxide-generating system which has been studied to date. The extent to which polyunsaturated fatty acid-dependent cooxygenation operates $\text{in vivo}$ is uncertain but preliminary studies suggest it does occur.