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

ABSTRACT

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 (GDH_Geo) (Fe[III] reducing) and Desulfococcus multivorans (GDH_Des) (sulfate reducing). GDH_Geo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDH_Des 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 GDH_Geo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDH_Des 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 GDH_Geo, the K_m was 30 ± 2 μM and the V _max was 3.2 ± 0.2 μmol min⁻¹ mg⁻¹, and for GDH_Des, the K_m was 52 ± 5 μM and the V _max was 11 ± 1 μmol min⁻¹ mg⁻¹. GDH_Des but not GDH_Geo 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 GDH_Geo but are missing in GDH_Des. 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.