Evolutionary divergence of nocturnin led to redox regulation in mammalian orthologs

NADP(H) and NAD(H) are essential cofactors involved in nearly 500 metabolic reactions, making them fundamental to cellular function and redox homeostasis. Loss of redox homeostasis through alterations in NADP(H) regulation has been implicated in various diseases, including neurodegenerative disorders and cardiovascular diseases. The levels of these metabolites are tightly regulated and highly compartmentalized. Nocturnin (NOCT), a circadian clock-controlled phosphatase, hydrolyzes both oxidized and reduced forms of NADP(H) and exists in cytosolic and mitochondrial forms. Here, we compare the activity of Xenopus and mammalian NOCT, which reveals a novel redox-dependent regulatory mechanism in mammals. Mammalian NOCT undergoes cysteine-mediated disulfide bond formation, leading to enzyme oligomerization and inactivation, while reduction of these cysteines restores NOCT to its monomeric, active form. In contrast, Xenopus laevis NOCT lacks these regulatory cysteines, does not oligomerize, and remains constitutively active. We show that oligomerization of mammalian NOCT occurs physiologically in the mitochondria while the comparatively highly reduced environment of the cytosol keeps NOCT in its monomeric conformation. Furthermore, sequence analysis reveals that mitochondrial NOCT emerges predominantly in mammals and coincides with the acquisition of redox-sensitive cysteines, highlighting a possible adaptation to oxidative metabolic environments. These findings suggest that mammalian NOCT functions as a redox sensor, dynamically modulating NADP(H) availability in response to oxidative stress. Understanding NOCT's evolution and regulation offers new insights into NADP(H) metabolism and its broader implications for oxidative stress.