Glutaryl‐coenzyme A dehydrogenase from Geobacter metallireducens – interaction with electron transferring flavoprotein and kinetic basis of unidirectional catalysis

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.

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