Abstract
Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP+ into NAD+ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Å crystal structure of the human NOCT•NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2′-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.
Highlights
Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock
NOCT is a member of the exonuclease/endonuclease/phosphatase family of proteins which include PDE12, a mitochondrial deadenylase needed for maturation of mitochondrial tRNAs, and CNOT6L, the mammalian ortholog of the main cytosolic yeast deadenylase CCR412,13
Based on the absence of mRNA deadenylase activity in purified NOCT, and its known role in metabolism, we hypothesized that NOCT could cleave a metabolite
Summary
Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. The NOCT−/− mice have low sensitivity to insulin and glucose, and have altered nitric oxide signaling, indicating a deep integration of this circadian protein in mammalian metabolism[6,9,10,11]. As a growing body of literature linked the biological effects of NOCT to mRNA deadenylation, two reports described the lack of deadenylase activity in highly purified NOCT in vitro[7,14]. We confirm the second model and show direct cleavage of two chemically related metabolites by human NOCT and by its fruit fly ortholog, Curled
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