title: Glutaryl-coenzyme A dehydrogenase from Geobacter metallireducens - interaction with electron transferring flavoprotein and kinetic basis of unidirectional catalysis.
abstract: Glutaryl-CoA dehydrogenases (GDHs) are FAD containing acyl-CoA dehydrogenases that usually catalyze the dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA with an electron transferring flavoprotein (ETF) acting as natural electron acceptor. In anaerobic bacteria, GDHs play an important role in the benzoyl-CoA degradation pathway of monocyclic aromatic compounds. In the present study, we identified, purified and characterized the benzoate-induced BamOP as the electron accepting ETF of GDH (BamM) from the Fe(III)-respiring Geobacter metallireducens. The BamOP heterodimer contained FAD and AMP as cofactors. In the absence of an artificial electron acceptor, at pH values above 8, the BamMOP-components catalyzed the expected glutaryl-CoA oxidation to crotonyl-CoA and CO2 ; however, at pH values below 7, the redox-neutral glutaryl-CoA conversion to butyryl-CoA and CO2 became the dominant reaction. This previously unknown, strictly ETF-dependent coupled glutaryl-CoA oxidation/crotonyl-CoA reduction activity was facilitated by an unexpected two-electron transfer between FAD(BamM) and FAD(BamOP) , as well as by the similar redox potentials of the two FAD cofactors in the substrate-bound state. The strict order of electron/proton transfer and C-C-cleavage events including transient charge-transfer complexes did not allow an energetic coupling of electron transfer and decarboxylation. This explains why it was difficult to release the glutaconyl-CoA intermediate from reduced GDH. Moreover, it provides a kinetic rational for the apparent inability of BamM to catalyze the reverse reductive crotonyl-CoA carboxylation, even under thermodynamically favourable conditions. For this reason reductive crotonyl-CoA carboxylation, a key reaction in C2-assimilation via the ethylmalonyl-CoA pathway, is accomplished by a different crotonyl-CoA carboxylase/reductase via a covalent NADPH/ene-adduct.
authors.affiliation: Institute of Biology II, University of Freiburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Estelmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Boll
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D044927:Electron-Transferring Flavoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D045302:Geobacter / Q000201:enzymology*; D050770:Glutaryl-CoA Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D044927:Electron-Transferring Flavoproteins; D050770:Glutaryl-CoA Dehydrogenase
keywords: FAD; Geobacter metallireducens; acyl-CoA dehydrogenase; electron transferring flavoprotein; glutaryl-CoA dehydrogenase