Abstract
NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a new substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we find that NADK2 is decorated with numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and are critical for mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal new molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.