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 nicotinamide mononucleotide (NMN) and AMP by SELENOO (SelO), using Mn²⁺ as cofactor. Catalysis depends on SelO's selenocysteine-serine-serine (CSS) C-terminal residues, particularly the selenocysteine 667. In addition to broad metabolic effects, this reaction plays a pronounced role in lipid utilization via SelO directly associating with fatty acid oxidation (FAO) enzymes, and it 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.