ADP-ribose is a competitive inhibitor of methanol dehydrogenases from Bacillus methanolicus.

Methanol dehydrogenase (MDH), a representative of Type III alcohol dehydrogenases (ADHs), plays a pivotal role in methanol assimilation pathways, making it a key enzyme for the biosynthesis of chemicals and fuels from one-carbon feedstocks. An activator protein belonging to the Nudix hydrolase family, ACT, was found to increase the activity of MDH by 40-fold. Despite the widespread observation of this in vitro activation phenomenon in pairs of type III alcohol dehydrogenases and Nudix hydrolases, the mechanistic details have remained unresolved for decades. Here, we uncover a regulation mechanism in which MDH activation arises from the hydrolytic removal of ADP-ribose (ADPR), a potent inhibitor derived from NAD+ degradation, by the ADPRase activity of ACT. This discovery challenges the previously proposed 'activation' models, revealing that ACT-mediated ADPR clearance disinhibits MDH rather than directly enhancing catalysis. By combining crystallographic analysis, kinetics, and inhibition assays, we demonstrate that ADPR inhibits MDHs with submicromolar Ki values, highlighting its potential regulatory role in metabolic networks. Our findings redefine the widespread 'activation' of type III ADHs, providing valuable insights into alcohol metabolism and new directions for engineering synthetic methanol utilization pathways.