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 (Km= 0.92 $\pm$ 0.15 $\mu$M), and an apparent specificity constant of 2.0 $\pm$ 0.3 $\mu$M-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.
Deep Dive into 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 (Km= 0.92 $\pm$ 0.15 $\mu$M), and an apparent specificity constant of 2.0 $\pm$ 0.3 $\mu$M-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 d
Ring-hydroxylating dioxygenases (RHDs) are widely spread bacterial enzymes that play a critical role in the biological degradation of a large array of aromatic compounds, including polycyclic aromatic hydrocarbons (PAHs) (1,2). RHDs catalyze the initial oxidation step of such compounds, which consists in the hydroxylation of two adjacent carbon atoms of the aromatic ring, thus generating a cis-dihydrodiol. This reaction converts hydrophobic, often toxic, molecules, into more hydrophilic products, allowing for their subsequent metabolism by other bacterial enzymes. Some RHDs were found to attack highly recalcitrant environmental pollutants, including dibenzo p-dioxin (3,4), polychlorobiphenyls (5), and PAHs (6)(7)(8), thus promoting studies on this type of enzymes with the ultimate goal of improving bioremediation processes (2,9). RHDs are multi-component enzymes, generally composed of a NADH-oxidoreductase, a ferredoxin and an oxygenase component that contains the active site. Sometimes, the reductase and the ferredoxin are fused in a single polypeptide. The oxygenase component is a multimeric protein, with either an n n (n=2 or 3) or 3 structure, that contains one [2Fe-2S] Rieske cluster and one non-heme iron atom per subunit (1). During a catalytic cycle, two electrons from the reduced pyridine nucleotide are transferred, via the reductase, the ferredoxin and the Rieske center, to the Fe(II) ion at the active site. The reducing equivalents allow the activation of molecular oxygen, which is a prerequisite to dihydroxylation of the substrate (10).
So far, only a few RHDs have been purified and extensively characterized, including phthalate dioxygenase (11,12), naphthalene dioxygenase (13,14) and biphenyl dioxygenase (15). None of these enzymes is able to oxidize substrates with more than three fused rings, and data on the mechanism, kinetics and efficiency of the oxidation of high molecular weight PAHs by bacterial dioxygenases are relatively scarce (16). However, the four-ring PAHs chrysene and benz[a]anthracene, and the five-ring benzo[a]pyrene are of particular concern because they are well-documented carcinogens (17). Recently, a Sphingomonad endowed with the remarkable ability to grow on chrysene as sole carbon and energy source was isolated in our laboratory (18). In this strain, called Sphingomonas sp. CHY-1, a single dioxygenase was shown to be responsible for the oxidation of polycyclic hydrocarbons made of 2 to 4 rings (6). In the present study, the three components of the dioxygenase were purified and characterized, and the catalytic properties of the enzyme with respect to the oxidation of nine PAHs were examined. Due to the broad specificity of this enzyme, the kinetics and coupling efficiency of the dioxygenase-catalyzed reaction with 2 to 5-ring PAHs could be compared for the first time. Steady-state kinetic parameters were determined for representative 2-ring PAHs. In addition, the reactivity and regioselectivity of the enzyme towards benz[a]anthracene was further investigated by means of single turnover chemistry and EPR spectroscopy.
Strains of Escherichia coli and Pseudomonas putida carrying the relevant expression plasmids, as well as general culture conditions, have been previously described (6). Largescale cultures required for the purification of the enzyme components were grown on rich medium, either Luria-Bertani or Terrific broth (19), in a 12-L fermentor (Discovery 100, SGI-Inceltech/New Brunswick Scientific, Paris, France). Cultures destined to the overproduction of the oxygenase or the ferredoxin component were supplemented with 50 µM ferrous ammonium sulfate. The medium was inoculated with 400 ml of an overnight culture, then incubated at 37°C under constant aeration and agitation (500 rpm), until the bacterial density (OD 600 ) reached about 1.0. The temperature was then lowered to 25°C, IPTG was added to 0.2 mM final concentration, and the culture was further incubated for 20 h before being harvested by centrifugation. The bacterial pellet was washed with 50 mM Tris-HCl buffer (pH 7.5), and kept frozen until use.
All purification procedures were carried out under argon, using buffers equilibrated for at least 24 h in a glove box maintained under anoxic conditions (O 2 <2 ppm, Jacomex , France).
The temperature was kept at 0-4 °C except when otherwise indicated. Crude extracts were prepared by thawing the bacterial pellets in twice as much lysis buffer by volume, followed by lysozyme treatment (0.5 mg/ml) for 15 min at 30°C. The lysis buffer was either 50 mM Tris-HCl, pH 7.5 (oxygenase preparation), 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10% glycerol (reductase preparation) or 50 mM potassium phosphate, pH 7.5, 0.5 M NaCl, 10% glycerol, 2 mM -mercaptoethanol (ferredoxin preparation). The suspension was then subjected to ultrasonication for a total time of 5 min at 80% of maximal intensity, using a Vibra Cell apparatus run in pulse mode at 5 s/pulse (Fisher Bioblock S
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