Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbons † (original) (raw)
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 µM), and an apparent specificity constant of 2.0 ± 0.3 µM-1 s-1. Naphthalene was converted to the corresponding 1,2dihydrodiol 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 ringdihydroxylation, giving rise to the formation of a significant proportion of bis-cisdihydrodiols. 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. 4 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 (6-8), 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