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Applied and Environmental Microbiology, November 2004, p. 6714-6725, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6714-6725.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

Sandrine Demanèche,1 Christine Meyer,1 Julien Micoud,1 Mathilde Louwagie,2 John C. Willison,1 and Yves Jouanneau1*

Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Unité Mixte de Recherche CEA-CNRS-Université Joseph Fourier-UMR5092,1 Laboratoire de Chimie des Protéines, Département de Réponse et Dynamique Cellulaires, CEA-Grenoble, Grenoble, France2

Received 29 April 2004/ Accepted 6 July 2004

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.

* Corresponding author. Mailing address: CEA-Grenoble, DRDC/BBSI, F-38054 Grenoble Cedex 9, France. Phone: 33 (0)4.38.78.43.10. Fax: 33 (0)4.38.78.51.85. E-mail: yjouanneau{at}cea.fr.

Applied and Environmental Microbiology, November 2004, p. 6714-6725, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6714-6725.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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