NADK2 encodes the mitochondrial NAD kinase 2, which serves as the primary enzyme responsible for generating mitochondrial NADP+ by phosphorylating NAD+ 1. This enzyme functions as a constitutively active dimer, structurally distinct from other NAD kinases that typically form tetramers 2. NADK2's primary mechanism involves producing mitochondrial NADPH, which is essential for several critical cellular processes. The enzyme is uniquely required for proline biosynthesis, as NADK2-deficient cells become proline auxotrophic due to impaired reduction of glutamate to pyrroline-5-carboxylate 13. Additionally, NADK2-derived NADPH fuels mitochondrial fatty acid synthesis and protein lipoylation, processes essential for optimal mitochondrial enzyme complex activity and oxidative metabolism 4. The enzyme's activity is regulated by post-translational modifications, including lysine acetylation at positions K76 and K304, which inhibit activity and reduce mitochondrial NADPH production 5. Disease-wise, NADK2 deficiency causes a rare neurological mitochondrial disorder characterized by hyperlysinemia and 2,4-dienoyl-CoA reductase deficiency 6. Mouse models demonstrate severe neuromuscular disease, cerebellar degeneration, and shortened lifespan, highlighting NADK2's critical role in neuronal function and metabolic homeostasis 67.