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Journal of Bacteriology, July 2009, p. 4401-4409, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00205-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria{triangledown}

Simon Wischgoll,1 Martin Taubert,1,3 Franziska Peters,2 Nico Jehmlich,3 Martin von Bergen,3 and Matthias Boll1*

Institute of Biochemistry, University of Leipzig, Leipzig, Germany,1 Institute of Biology II, University of Freiburg, Freiburg, Germany,2 Helmholtz Centre for Environmental Research—UFZ, Department of Proteomics, Leipzig, Germany3

Received 16 February 2009/ Accepted 19 April 2009

In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO2. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDHGeo) (Fe[III] reducing) and Desulfococcus multivorans (GDHDes) (sulfate reducing). GDHGeo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDHDes was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDHGeo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDHDes catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDHGeo, the Km was 30 ± 2 µM and the Vmax was 3.2 ± 0.2 µmol min–1 mg–1, and for GDHDes, the Km was 52 ± 5 µM and the Vmax was 11 ± 1 µmol min–1 mg–1. GDHDes but not GDHGeo was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDHGeo but are missing in GDHDes. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

* Corresponding author. Mailing address: Institute of Biochemistry, University of Leipzig, Brüderstr. 34, D-04103 Leipzig, Germany. Phone: 49-341-9736996. Fax: 49-341-9736910. E-mail: boll{at}uni-leipzig.de

{triangledown} Published ahead of print on 24 April 2009.

Journal of Bacteriology, July 2009, p. 4401-4409, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00205-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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