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Biochemistry. 2006 Oct 10;45(40):12380-91.

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

Author information

  • 1CEA, DSV, DRDC, Lab. Biochim. Biophys. Syst. Intégrés, CNRS, UMR 5092, F-38054 Grenoble, France. yves.jouanneau@cea.fr

Abstract

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.

PMID:
17014090
[PubMed - indexed for MEDLINE]
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