Dual feedback inhibition of ATP-dependent caffeate activation economizes ATP in caffeate-dependent electron bifurcation

The acetogen Acetobacterium woodii couples caffeate reduction with ferredoxin reduction and NADH oxidation via electron bifurcation, providing additional reduced ferredoxin for energy conservation and cell synthesis. Caffeate is first activated by an acyl-CoA synthetase (CarB), which ligates CoA to caffeate at the expense of ATP. After caffeoyl-CoA is reduced to hydrocaffeoyl-CoA, the CoA moiety in hydrocaffeoyl-CoA could be recycled for caffeoyl-CoA synthesis by an ATP-independent CoA transferase (CarA) to save energy. However, given that CarA and CarB are co-expressed, it was not well understood how ATP could be saved when both two competitive pathways of caffeate activation are present. Here, we reported a dual feedback inhibition of the CarB-mediated caffeate activation by the intermediate hydrocaffeoyl-CoA and the end-product hydrocaffeate. As the product of CarA, hydrocaffeate inhibited CarB-mediated caffeate activation by serving as another substrate of CarB with hydrocaffeoyl-CoA produced. It effectively competed with caffeate even at a concentration much lower than caffeate. Hydrocaffeoyl-CoA formed in this process can also inhibit CarB-mediated caffeate activation. Thus, the dual feedback inhibition of CarB, together with the faster kinetics of CarA, makes the ATP-independent CarA-mediated CoA loop the major route for caffeoyl-CoA synthesis, further saving ATP in the caffeate-dependent electron-bifurcating pathway. A genetic architecture similar to carABC has been found in other anaerobic bacteria, suggesting that the feedback inhibition of acyl-CoA ligases could be a widely employed strategy for ATP conservation in those pathways requiring substrate activation by CoA.

Importance: This study reports a dual feedback inhibition of caffeoyl-CoA synthetase by two downstream products, hydrocaffeate and hydrocaffeoyl-CoA. It elucidates how such dual feedback inhibition suppresses ATP-dependent caffeoyl-CoA synthesis, hence making the ATP-independent route the main pathway of caffeate activation. This newly discovered mechanism contributes to our current understanding of ATP conservation during the caffeate-dependent electron-bifurcating pathway in the ecologically important acetogen Acetobacterium woodii. Bioinformatic mining of microbial genomes revealed contiguous genes homologous to carABC within the genomes of other anaerobes from various environments, suggesting this mechanism may be widely used in other CoA-dependent electron-bifurcating pathways.