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Applied and Environmental Microbiology, December 2007, p. 7515-7521, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01519-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from Sphingomonas sp. Strain CHY-1{triangledown}

Yves Jouanneau,* Julien Micoud, and Christine Meyer

CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, 17 rue des Martyrs, F-38054 Grenoble, CNRS, UMR 5249, F-38054 Grenoble, and Université Joseph Fourier, F-38000 Grenoble, France

Received 5 July 2007/ Accepted 19 September 2007

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an {alpha}3ß3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per {alpha} subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent Km for salicylate of 1.1 ± 0.2 µM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (Km, 28 ± 6 µM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a Ki of 0.68 µM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.

* Corresponding author. Mailing address: LCBM/iRTSV, CEA-Grenoble, F-38054 Grenoble Cedex 9, France. Phone: 33 (0)4 38 78.43.10. Fax: 33 (0)4 38 78.51.85. E-mail: yves.jouanneau{at}cea.fr

{triangledown} Published ahead of print on 28 September 2007.

Applied and Environmental Microbiology, December 2007, p. 7515-7521, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01519-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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