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Title: Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons

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{sub m} = 0.92 {+-} 0.15 {mu}M) and an apparent specificity constant of 2.0 {+-} 0.3 M{sup -1} s{sup -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,more » 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.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930083
Report Number(s):
BNL-80716-2008-JA
Journal ID: ISSN 0006-2960; TRN: US200822%%1299
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 45
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CHRYSENE; COUPLING; ELECTRON SPIN RESONANCE; ENZYMES; HYDROGEN PEROXIDE; HYDROXYLATION; IRON; KINETICS; NAPHTHALENE; ORGANIC SOLVENTS; OXIDATION; OXYGENASES; POLYCYCLIC AROMATIC HYDROCARBONS; RANGE; REACTION KINETICS; SPECIFICITY; SUBSTRATES; national synchrotron light source

Citation Formats

Jouanneau,Y., Meyer, C., Jakoncic, J., Stojanoff, V., and Gaillard, J. Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons. United States: N. p., 2006. Web. doi:10.1021/bi0611311.
Jouanneau,Y., Meyer, C., Jakoncic, J., Stojanoff, V., & Gaillard, J. Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons. United States. doi:10.1021/bi0611311.
Jouanneau,Y., Meyer, C., Jakoncic, J., Stojanoff, V., and Gaillard, J. Sun . "Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons". United States. doi:10.1021/bi0611311.
@article{osti_930083,
title = {Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons},
author = {Jouanneau,Y. and Meyer, C. and Jakoncic, J. and Stojanoff, V. and Gaillard, J.},
abstractNote = {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{sub m} = 0.92 {+-} 0.15 {mu}M) and an apparent specificity constant of 2.0 {+-} 0.3 M{sup -1} s{sup -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.},
doi = {10.1021/bi0611311},
journal = {Biochemistry},
number = ,
volume = 45,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}