NAD+ Hydrolysis Catalyzed by SelO is Required for Mitochondrial Homeostasis

The regulation of nicotinamide adenine dinucleotide (NAD+) is crucial for numerous life processes. However, the mechanisms leading to NAD+ degradation in mitochondria remain insufficiently defined. Through in silico screening of potential NAD-binding proteins, we discovered a mitochondrial reaction in which NAD+ is hydrolyzed to NMN and AMP by SelO, using Mn2+ as cofactor. Catalysis depends on SelO’s CSS c-terminal residues, particularly the selenocysteine 667. In addition to broad metabolic effects, this reaction exerts a pronounced role on lipid utilization via SelO directly associating with FAO enzymes, which is conserved in both mammalian cells and bacteria. This reaction is responsive to elevated matrix pH, a signal of enhanced mitochondrial respiration, and protects mitochondria from sustained metabolic overactivation. These findings reveal a conserved mechanism for spatiotemporal NAD+ regulation, and highlight its physiological significance in both prokaryotes and eukaryotes.