Investigating the Origin of Substrate Specificity in the Circadian NADP(H) Phosphatase Nocturnin

Abstract

Nocturnin is a recently discovered gene that displays one of the highest-amplitude rhythmic expressions known and is expressed in many tissues of various organisms. Mice with targeted disruption of Nocturnin (NOCT) exhibit resistance to diet-induced obesity and hepatic steatosis. Originally, it has been proposed that NOCT deadenylates metabolic enzyme mRNAs as it has been found that NOCT has a close similarity to CCR4 deadenylase family members CNOT6L (the major deadenylase in yeast cytosol) and PDE12 (the major deadenylase in mammalian mitochondria). Indeed, all key catalytic residues present in CNOT6L and PDE12 are conserved in NOCT and have the same conformations. However, our lab recently refuted this model. We discovered that NOCT is a metabolic enzyme that does not cleave poly-A RNA, but instead converts NADP(H) to NAD(H) under control of the circadian clock. This study aims to determine the precise set of residues responsible for the substrate specificity of NOCT. Toward this end, I am using structure-guided mutagenesis of the protein accompanied by testing with biochemical assays. We expected that a switch of substrate specificity from NADP(H) to poly-A RNA could be achieved through mutations in the central active site based on differences between CNOT6L/PDE12 and NOCT. However, mutation of central active site residues was not sufficient for engineering deadenylase activity in NOCT, suggesting that the mechanism is considerably more interesting and mysterious. Future studies examining remote active site residues and global protein surface property differences should be completed in order to better understand the substrate specificity of NOCT.