Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbons
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- 作者：Yves Jouanneau ; Christine Meyer ; Jean Jakoncic ; Vivian Stojanoff ; Jacques Gaillard
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：October 10, 2006
- 年：2006
- 卷：45
- 期：40
- 页码：12380 - 12391
- 全文大小：122K
- 年卷期：v.45,no.40(October 10, 2006)
- ISSN：1520-4995
文摘
In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initialattack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The componentsof this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shownto contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per 
subunit, based on EPRmeasurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relativelylow apparent Michaelis constant for naphthalene (Km = 0.92 ± 0.15 
M) and an apparent specificityconstant of 2.0 ± 0.3 M-1 s-1. Naphthalene was converted to the corresponding 1,2-dihydrodiol withstoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred atslower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling wasassociated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibitPAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a fasterrate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is alimiting 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 preferencefor carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able todihydroxylate PAHs made up of four and five rings.
subunit, based on EPRmeasurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relativelylow apparent Michaelis constant for naphthalene (Km = 0.92 ± 0.15 M) and an apparent specificityconstant of 2.0 ± 0.3 M-1 s-1. Naphthalene was converted to the corresponding 1,2-dihydrodiol withstoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred atslower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling wasassociated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibitPAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a fasterrate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is alimiting 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 preferencefor carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able todihydroxylate PAHs made up of four and five rings.