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Journal of Bacteriology, March 2001, p. 1899-1908, Vol. 183, No. 6
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.6.1899-1908.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Reinvestigation of a New Type of Aerobic Benzoate Metabolism in the Proteobacterium Azoarcus evansii

Magdy El-Said Mohamed,dagger Annette Zaar, Christa Ebenau-Jehle, and Georg Fuchs*

Mikrobiologie, Institut Biologie II, Universität Freiburg, Freiburg, Germany

Received 25 May 2000/Accepted 4 December 2000

The aerobic metabolism of benzoate in the proteobacterium Azoarcus evansii was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of benzoate to benzoyl-CoA by a specifically induced benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.72 mol flavin adenine dinucleotide (FAD), 10.4 to 18.4 mol of Fe, and 13.3 to 17.9 mol of acid-labile sulfur per mol of native protein, depending on the method of protein determination. This benzoate-induced enzyme catalyzed a benzoyl-CoA-, FAD-, and O2-dependent NADPH oxidation surprisingly without hydroxylation of the aromatic ring; however, H2O2 was formed. The gene (boxA, for benzoate oxidation) coding for this protein was cloned and sequenced. It coded for a protein of 46 kDa with two amino acid consensus sequences for two [4Fe-4S] centers at the N terminus. The deduced amino acid sequence showed homology with subunits of ferredoxin-NADP reductase, nitric oxide synthase, NADPH-cytochrome P450 reductase, and phenol hydroxylase. Upstream of the boxA gene, another gene, boxB, encoding a protein of 55 kDa was found. The boxB gene exhibited homology to open reading frames in various other bacteria which code for components of a putative aerobic phenylacetyl-CoA oxidizing system. The boxB gene product was one of at least five proteins induced when A. evansii was grown on benzoate.

* Corresponding author. Mailing address: Mikrobiologie, Institut Biologie II, Schänzlestr. 1, D-79104 Freiburg, Germany. Phone: 49-761-2032649. Fax: 49-761-2032626. E-mail: fuchsgeo{at}uni-freiburg.de.

dagger Present address: Botany Department, Faculty of Science, Cairo University-Giza, Giza, Egypt.

Journal of Bacteriology, March 2001, p. 1899-1908, Vol. 183, No. 6
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.6.1899-1908.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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