Isolation and characterization of Indigenous Bacilli strains from an oil refinery wastewater with potential applications for phenol/cresol bioremediation.

Phenolic compounds are frequently occurring in wastewaters from various industrial processes at high concentrations, imposing prominent risk to aquatic biosphere and human health. Bioremediation has been proven to be an effective approach to remove these compounds, and hunting for functional organisms is still of primary importance to develop efficient processes. In this study, we report several newly isolated bacillus strains with superior performances in metabolizing phenols, one of which showed paramount efficiencies to metabolize phenol at concentrations up to 1200 mg L-1 and could simultaneously degrade a wide range of other phenolic compounds. The genes encoding for phenol hydroxylase (PH) and catechol-2,3-dioxygenase (C23O) have been detected and characterized, evidencing that phenol degradation occurs via the meta pathway. The GC level of the PH gene was found to be much higher than that of genes from other Bacilli but was quite close to that of the genes from Rhodococcus, and the induction of both enzymes by phenols was confirmed by RT-PCR experiments. We intend to believe this novel strain might be promising to serve as preferred organisms for developing more robust and efficient bioremediation processes of degrading phenolic compounds due to its validated performance.

Diversity shift in bacterial phenol hydroxylases driven by alkyl-phenols in oil refinery wastewaters.

Phenol hydroxylases (PHs) play a primary role in the bacterial degradation of phenol and alkylphenols. They are divided into two main classes, single-component and multi-component PHs, having distinctive catalytic subunits designated as PheA1 and LmPH, respectively. The diversity of these enzymes is still largely unexplored. Here, both LmPH and pheA1 gene sequences were examined in activated sludge from oil refinery wastewaters. Phenol, p-cresol, or 3,4-dimethylphenol (3,4-DMP) supplied as extra carbon sources were rapidly mineralized by the microbial community. Analysis of LmPH genes revealed a wide range of sequences, most of which exhibited moderate similarity with homologs found in Proteobacteria. Moreover, the LmPH diversity profiles showed a dramatic shift upon sludge treatment with p-cresol or 3,4-DMP amendment. This resulted in an enrichment in sequences similar to LmPHs from Betaproteobacteria and Gammaproteobacteria. RT-PCR analysis of RNA extracted from wastewater sludge highlighted LmPH genes best expressed in situ. A PCR approach was implemented to analyze the pheA1 gene diversity in the same microbial community. Retrieved sequences fell into four clusters and appeared to be distantly related to pheA1 genes from Actinobacteria. Altogether, our results provide evidence that phenol degraders carrying LmPH are more diverse than PheA1 carrying bacteria and suggest that PHs with best adapted substrate specificity are recruited in response to (methyl)phenol availability.

Dihydroxylation of four- and five-ring aromatic hydrocarbons by the naphthalene dioxygenase from Sphingomonas CHY-1.

The naphthalene dioxygenase from Sphingomonas CHY-1 exhibits extremely broad substrate specificity toward polycyclic aromatic hydrocarbons (PAHs). In a previous study, the catalytic rates of oxidation of nine PAHs were determined using the purified dioxygenase, but the oxidation products formed from four- to five-ring hydrocarbons were incompletely characterized. Here, we reexamined PAH oxygenation reactions using Escherichia coli recombinant cells overproducing strain CHY-1 dioxygenase. Hydroxylated products generated by the dioxygenase were purified and characterized by means of GC-MS, UV absorbance as well as 1H- and 13C-NMR spectroscopy. Fluoranthene was converted to three dihydrodiols, the most abundant of which was identified as cis-7,8-dihydroxy-7,8-dihydrofluoranthene. This diol turned out to be highly unstable, converting to 8-hydroxyfluoranthene by spontaneous dehydration. The dioxygenase also catalyzed dihydroxylations on the C2-C3 and presumably the C1-C2 positions, although at much lower rates. Benz[a]anthracene was converted into three dihydrodiols, hydroxylated in positions C1-C2, C8-C9, and C10-C11, and one bis-cis-dihydrodiol. The latter compound was identified as cis,cis-1,2,10,11-tetrahydroxy-1,2,10,11-tetrahydrobenz[a]anthracene, which resulted from the subsequent dioxygenation of the 1,2- or 10,11-dihydrodiols. Chrysene dioxygenation yielded a single diol identified as cis-3,4-dihydroxy-3,4-dihydrochrysene, which underwent further oxidation to give cis,cis-3,4,9,10 chrysene tetraol. Pyrene was a poor substrate for the CHY-1 dioxygenase and gave a single dihydrodiol hydroxylated on C4 and C5, whereas benzo[a}pyrene was converted to two dihydrodiols, one of which was identified as cis-9,10-dihydrodiol. The selectivity of the dioxygenase is discussed in the light of the known 3D structure of its catalytic component and compared to that of the few enzymes able to attack four- and five-ring PAHs.

Characterization of novel polycyclic aromatic hydrocarbon dioxygenases from the bacterial metagenomic DNA of a contaminated soil.

Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [(13)C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.

Identification of soil bacteria able to degrade phenanthrene bound to a hydrophobic sorbent in situ.

Efficient bioremediation of PAH-contaminated sites is limited by the hydrophobic character and poor bioavailability of pollutants. In this study, stable isotope probing (SIP) was implemented to track bacteria that can degrade PAHs adsorbed on hydrophobic sorbents. Temperate and tropical soils were incubated with (13)C-labeled phenanthrene, supplied by spiking or coated onto membranes. Phenanthrene mineralization was faster in microcosms with PAH-coated membranes than in microcosms containing spiked soil. Upon incubation with temperate soil, phenanthrene degraders found in the biofilms that formed on coated membranes were mainly identified as Sphingomonadaceae and Actinobacteria. In the tropical soil, uncultured Rhodocyclaceae dominated degraders bound to membranes. Accordingly, ring-hydroxylating dioxygenase sequences recovered from this soil matched PAH-specific dioxygenase genes recently found in Rhodocyclaceae. Hence, our SIP approach allowed the detection of novel degraders, mostly uncultured, which differ from those detected after soil spiking, but might play a key role in the bioremediation of PAH-polluted soils.

Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil.

Ring-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes. Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria.

Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR.

BACKGROUND: Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr. RESULTS: Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]2+ cluster per monomer. Atmospheric O2 causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has a higher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex. CONCLUSIONS: Overall, this work shows that incorporation of the [4Fe-4S]2+ cluster is not required for DNA binding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.

Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene.

In this study, the PAH-degrading bacteria of a constructed wetland collecting road runoff has been studied through DNA stable isotope probing. Microcosms were spiked with (13)C-phenanthrene at 34 or 337 ppm, and bacterial diversity was monitored over a 14-day period. At 337 ppm, PAH degraders became dominated after 5 days by Betaproteobacteria, including novel Acidovorax, Rhodoferax and Hydrogenophaga members, and unknown bacteria related to Rhodocyclaceae. The prevalence of Betaproteobacteria was further demonstrated by phylum-specific quantitative PCR, and was correlated with a burst of phenanthrene mineralization. Striking shifts in the population of degraders were observed after most of the phenanthrene had been removed. Soil exposed to 34 ppm phenanthrene showed a similar population of degraders, albeit only after 14 days. Results demonstrate that specific Betaproteobacteria are involved in the main response to soil PAH contamination, and illustrate the potential of SIP approaches to investigate PAH biodegradation in soil.

Proteomic investigation of enzymes involved in 2-ethylhexyl nitrate biodegradation in Mycobacterium austroafricanum IFP 2173.

2-Ethyhexyl nitrate (2-EHN) is a synthetic chemical used as a diesel fuel additive, which is recalcitrant to biodegradation. In this study, the enzymes involved in 2-EHN degradation were investigated in Mycobacterium austroafricanum IFP 2173. Using two-dimensional gel electrophoresis and a shotgun proteomic approach, a total of 398 proteins appeared to be more abundant in cells exposed to 2-EHN than in acetate-grown cells. This set of proteins includes multiple isoenzymes of the beta-oxidation pathway, two alcohol and one aldehyde dehydrogenase, as well as four cytochromes P450, including one CYP153 which functions as an alkane hydroxylase. Strain IFP 2173 was also found to contain two alkB-like genes encoding putative membrane-bound alkane hydroxylases. RT-PCR experiments showed that the gene encoding the CYP153 protein, as well as alkB genes, were expressed on 2-EHN. These findings are discussed in the light of a recently proposed 2-EHN degradation pathway involving an initial attack by an alkane hydroxylase and one turn of beta-oxidation, leading to the accumulation of a gamma-lactone as a dead-end product.

Characterization of a ring-hydroxylating dioxygenase from phenanthrene-degrading Sphingomonas sp. strain LH128 able to oxidize benz[a]anthracene.

Sphingomonas sp. strain LH128 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated soil using phenanthrene as the sole source of carbon and energy. A dioxygenase complex, phnA1fA2f, encoding the alpha and beta subunit of a terminal dioxygenase responsible for the initial attack on PAHs, was identified and isolated from this strain. PhnA1f showed 98%, 78%, and 78% identity to the alpha subunit of PAH dioxygenase from Novosphingobium aromaticivorans strain F199, Sphingomonas sp. strain CHY-1, and Sphingobium yanoikuyae strain B1, respectively. When overexpressed in Escherichia coli, PhnA1fA2f was able to oxidize low-molecular-weight PAHs, chlorinated biphenyls, dibenzo-p-dioxin, and the high-molecular-weight PAHs benz[a]anthracene, chrysene, and pyrene. The action of PhnA1fA2f on benz[a]anthracene produced two benz[a]anthracene dihydrodiols.

Biodegradability of 2-ethylhexyl nitrate (2-EHN), a cetane improver of diesel oil.

The 2-ethyhexyl nitrate (2-EHN) is currently added to diesel oil to improve ignition and boost cetane number. The biodegradability of this widely used chemical needed to be assessed in order to evaluate the environmental impact in case of accidental release. In aerobic liquid cultures, biodegradation of 2-EHN was assessed in biphasic liquid cultures using an inert non-aqueous phase liquid such as 2,2,4,4,6,8,8-heptamethylnonane (HMN) as solvent for the hydrophobic substrate. 2-EHN was found to be biodegradable by microbial communities from refinery wastewater treatment plants, but was recalcitrant to those of urban wastewater treatment facilities. Out of eighteen hydrocarbon-polluted or non-polluted soil samples, six microbial populations were also able to degrade 2-EHN. However, strain isolation from these microbial populations was rather difficult suggesting close cooperation between members of the microbial communities. Specific axenic bacterial strains selected for their ability to catabolize recalcitrant-hydrocarbons were also tested for their capacity to degrade 2-EHN. In liquid cultures with HMN phase as non-aqueous phase liquid, some Mycobacterium austroafricanum strains were found to degrade and mineralize 2-EHN significantly.

Biodegradation of 2-ethylhexyl nitrate by Mycobacterium austroafricanum IFP 2173.

2-Ethyhexyl nitrate (2-EHN) is a major additive of fuel that is used to increase the cetane number of diesel. Because of its wide use and possible accidental release, 2-EHN is a potential pollutant of the environment. In this study, Mycobacterium austroafricanum IFP 2173 was selected from among several strains as the best 2-EHN degrader. The 2-EHN biodegradation rate was increased in biphasic cultures where the hydrocarbon was dissolved in an inert non-aqueous-phase liquid, suggesting that the transfer of the hydrophobic substrate to the cells was a growth-limiting factor. Carbon balance calculation, as well as organic-carbon measurement, indicated a release of metabolites in the culture medium. Further analysis by gas chromatography revealed that a single metabolite accumulated during growth. This metabolite had a molecular mass of 114 Da as determined by gas chromatography/mass spectrometry and was provisionally identified as 4-ethyldihydrofuran-2(3H)-one by liquid chromatography-tandem mass spectrometry analysis. Identification was confirmed by analysis of the chemically synthesized lactone. Based on these results, a plausible catabolic pathway is proposed whereby 2-EHN is converted to 4-ethyldihydrofuran-2(3H)-one, which cannot be metabolized further by strain IFP 2173. This putative pathway provides an explanation for the low energetic efficiency of 2-EHN degradation and its poor biodegradability.

ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.

Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (Fnr(His)) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) ((Strep)Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnr(His) was mainly monomeric, while apo(Strep)Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apo(Strep)Fnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

Characterization of a novel angular dioxygenase from fluorene-degrading Sphingomonas sp. strain LB126.

In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The alpha and beta subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in Escherichia coli, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-p-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2.

Purification and characterization of a three-component salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1.

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an alpha3beta3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per alpha subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent Km for salicylate of 1.1 +/- 0.2 microM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (Km, 28 +/- 6 microM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a Ki of 0.68 microM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.

The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1.

The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 A by molecular replacement revealed the enzyme to share the same global alpha(3)beta(3) structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 A. An 11 A long water channel extends from the interface between the alpha and beta subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.

The catalytic pocket of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1.

Ring-hydroxylating dioxygenases are multicomponent bacterial enzymes that catalyze the first step in the oxidative degradation of aromatic hydrocarbons. The dioxygenase from Sphingomonas CHY-1 is unique in that it can oxidize a wide range of polycyclic aromatic hydrocarbons (PAHs). With a crystal structure similar to that of the seven other known dioxygenases, its catalytic domain features the largest hydrophobic substrate binding cavity characterized so far. Molecular modeling studies indicated that the catalytic cavity is large enough to accommodate a five-ring benzo[a]pyrene molecule. The predicted positions of this and other PAHs in the substrate binding pocket are consistent with the product regio- and stereo-selectivity of the enzyme.

Characterization of a naphthalene dioxygenase endowed with an exceptionally broad substrate specificity toward polycyclic aromatic hydrocarbons.

In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per alpha subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K(m) = 0.92 +/- 0.15 microM) and an apparent specificity constant of 2.0 +/- 0.3 mM(-)(1) s(-)(1). Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings.

Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols.

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyrene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 microM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 microM). At pH 7.0, the specificity constant ranged from (1.3 +/- 0.1) x 10(6) M(-1) s(-1) with benz[a]anthracene 1,2-dihydrodiol to (20.0 +/- 0.8) x 10(6) M(-1) s(-1) with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.

Structure of a [2Fe-2S] ferredoxin from Rhodobacter capsulatus likely involved in Fe-S cluster biogenesis and conformational changes observed upon reduction.

FdVI from Rhodobacter capsulatus is structurally related to a group of [2Fe-2S] ferredoxins involved in iron-sulfur cluster biosynthesis. Comparative genomics suggested that FdVI and orthologs found in alpha-Proteobacteria are involved in this process. Here, the crystal structure of FdVI has been determined for both the oxidized and the reduced protein. The [2Fe-2S] cluster lies 6 A below the protein surface in a hydrophobic pocket without access to the solvent. This particular cluster environment might explain why the FdVI midpoint redox potential (-306 mV at pH 8.0) did not show temperature or ionic strength dependence. Besides the four cysteines that bind the cluster, FdVI features an extra cysteine which is located close to the S1 atom of the cluster and is oriented in a position such that its thiol group points towards the solvent. Upon reduction, the general fold of the polypeptide chain was almost unchanged. The [2Fe-2S] cluster underwent a conformational change from a planar to a distorted lozenge. In the vicinity of the cluster, the side chain of Met24 was rotated by 180 degrees , bringing its S atom within hydrogen-bonding distance of the S2 atom of the cluster. The reduced molecule also featured a higher content of bound water molecules, and more extensive hydrogen-bonding networks compared with the oxidized molecule. The unique conformational changes observed in FdVI upon reduction are discussed in the light of structural studies performed on related ferredoxins.