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Applied and Environmental Microbiology, July 2006, p. 4726-4734, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00395-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols

Yves Jouanneau^* and Christine Meyer

CEA, DSV, Département de Réponse et Dynamique Cellulaires/BBSI and CNRS UMR 5092, CEA-Grenoble, F-38054 Grenoble Cedex 9, France

Received 17 February 2006/ Accepted 24 April 2006

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent M_r of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with K_m values in the range of 1.4 to 7.1 µM, and exhibited a much higher Michaelis constant for NAD⁺ (K_m of 160 µM). At pH 7.0, the specificity constant ranged from (1.3 ± 0.1) x 10⁶ M^–1 s^–1 with benz[a]anthracene 1,2-dihydrodiol to (20.0 ± 0.8) x 10⁶ M^–1 s^–1 with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.

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* Corresponding author. Mailing address: Département de Réponse et Dynamique Cellulaires/BBSI, CEA-Grenoble, 17 avenue des Martyrs, F-38054 Grenoble Cedex 9, France. Phone: 33 4 38 78 43 10. Fax: 33 4 38 78 51 85. E-mail: yves.jouanneau{at}cea.fr.

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Applied and Environmental Microbiology, July 2006, p. 4726-4734, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00395-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Jouanneau, Y., Micoud, J., Meyer, C. (2007). Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from Sphingomonas sp. Strain CHY-1. Appl. Environ. Microbiol. 73: 7515-7521 [Abstract] [Full Text]