AbstractClass-II fumarases (Fumarate Hydratase, FH) are dual targeted enzymes, occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and more specifically, protecting cells from DNA double strand breaks. Similarly, the Gram-positive Bacterium Bacillus subtilis Class-II fumarase, in addition to its role in the TCA cycle, also participates in the DDR. Escherichia coli, harbors three fumarase genes; Class-I fumA and fumB and Class-II fumC. Notably, Class-I fumarases, show no sequence similarity to Class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between Class-I fumarases which participate in the DDR, and the Class-II fumarase which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a novel function, complementing DNA damage sensitivity of fum null mutants. Excitingly, we identify the E. coli α-KG dependent DNA repair enzyme AlkB, as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, as fum null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together these results show evolutionary adaptable metabolic signaling of the DDR in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme Class preforming the function.Significance StatementClass-II fumarases have been shown to participate in cellular respiration and the DNA damage response. Here we show, for the first time, that in the model prokaryote,Escherichia coli, which harbors both Class-I and Class-II fumarases, it is the Class-I fumarases that participate in DNA damage repair by a mechanism which is different than those described for other fumarases. Strikingly, this mechanism employs a novel signaling molecule, alpha-ketoglutarate (α-KG), and its target is the DNA damage repair enzyme AlkB. In addition, we show that fumarase precursor metabolites, fumarate and succinate, can inhibit the α-KG-dependent DNA damage repair enzyme, AlkB, both in vitro and in vivo. This study provides a new perspective on the function and evolution of metabolic signaling.
DNA Damage Repair and Drug Efflux as Potential Targets for Reversing Low or Intermediate Ciprofloxacin Resistance in E. coli K-12
