biostudies-literatureKim DU.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)V Foundation for Cancer Research (V Foundation)U.S. Department of Health & Human Services | NIH | National Institute of General Medical SciencesU.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)Pew Charitable TrustsU.S. Department of Health & Human Services | National Institutes of Health (NIH)U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)Cancer Prevention and Research Institute of TexasUnited States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)American Cancer SocietyU.S. Department of Health & Human Services | NIH | National Cancer InstituteAmerican Cancer Society (American Cancer Society, Inc.)NCI NIH HHSWelch FoundationUnited States Department of Agriculture | Agricultural Research ServiceV Foundation for Cancer ResearchNIGMS NIH HHSU.S. Department of Health & Human Services | National Institutes of HealthU.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases790-800https://www.ebi.ac.uk/biostudies/studies/S-EPMC12331256biostudies-literatureUnknown27(5)Nicotinamide adenine dinucleotide phosphate (NADPH) is a vital electron donor essential for macromolecular biosynthesis and protection against oxidative stress. Although NADPH is compartmentalized within the cytosol and mitochondria, the specific functions of mitochondrial NADPH remain largely unexplored. Here we demonstrate that NAD⁺ kinase 2 (NADK2), the principal enzyme responsible for mitochondrial NADPH production, is critical for maintaining protein lipoylation, a conserved lipid modification necessary for the optimal activity of multiple mitochondrial enzyme complexes, including the pyruvate dehydrogenase complex. The mitochondrial fatty acid synthesis (mtFAS) pathway utilizes NADPH for generating protein-bound acyl groups, including lipoic acid. By developing a mass-specNature cell biologyMitochondrial NADPH fuels mitochondrial fatty acid synthesis and lipoylation to power oxidative metabolism.PMC12331256V2021-019DK1374071R01CA289251-01A1R01 CA2892513R01GM143236-02S1S10OD030312-012021-67013-3377800036920RR190087R01 GM143236RP210041RSG-22-177-01-TBERP240035R01GM143236I-2067-20240404Kim DDey TSoflaee MHMishra PRyu KMenezes CKo BBrautigam CALemoff AAllen DKBrown HCai FKesavan RHoxhaj GTom HJMarckx AMettlen MMorley SPraharaj PPVu HSTso SCfalseMitochondrial NADPH fuels mitochondrial fatty acid synthesis and lipoylation to power oxidative metabolism.Nicotinamide adenine dinucleotide phosphate (NADPH) is a vital electron donor essential for macromolecular biosynthesis and protection against oxidative stress. Although NADPH is compartmentalized within the cytosol and mitochondria, the specific functions of mitochondrial NADPH remain largely unexplored. Here we demonstrate that NAD⁺ kinase 2 (NADK2), the principal enzyme responsible for mitochondrial NADPH production, is critical for maintaining protein lipoylation, a conserved lipid modification necessary for the optimal activity of multiple mitochondrial enzyme complexes, including the pyruvate dehydrogenase complex. The mitochondrial fatty acid synthesis (mtFAS) pathway utilizes NADPH for generating protein-bound acyl groups, including lipoic acid. By developing a mass-spec2025-01-01T00:00:00Z2025 May2026-04-07T13:35:56.752Z2026-04-07T13:28:52.601ZS-EPMC123312564025894910.1038/s41556-025-01655-4