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Journal of Bacteriology, March 2001, p. 1899-1908, Vol. 183, No. 6
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.
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
*
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.
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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