Nocturnin is an enzyme belonging to the EEP (endonuclease/exonuclease/phosphatase) structural family that is expressed rhythmically under the control of the circadian oscilator. It has been hypothesized to control the stability of other circadian clock controlled transcripts through degradation of their 3' poly(A) tails, and shows deadenylase activity in vitro. Functional studies in cell culture and kockout mice have implicated nocturnin in the regulation of intestinal lipid absorption, dipocyte and osteoblast formation, and lipid and glucose metabolism in hepatocytes. We have discovered that nocturnin's hydrolytic activity is not limited to deadenylation, but it can also act as a phosphatase and dephosphorylate AMP and other substrates, raising the question as to which activity is responsible for its primary cellular function. As nocturnin represents an important target for understanding and modulating the interactions between circadian rhythms and metabolism in homeostasis and disease, the experiments described in this proposal are designed to characterize the steady state kinetics, substrate specificity, and three dimensional structure of nocturnin.