Characterization of the Human Lipin 1 Phosphatidate Phosphatase Activity

Lipin 1 plays an important role in fat metabolism in mammalian cells. Its deficiency causes lipodystrophy, whereas its excess causes obesity. Recent studies identified the molecular function of lipin 1 as a Mg 2+ ‐dependent phosphatidate (PA) phosphatase enzyme. In this work, we examined the enzymological properties of His 6 ‐tagged human lipin 1 expressed in Escherichia coli . The PA phosphatase activity of lipin 1 was dependent on magnesium ions with optimal activity at 0.5 mM MgCl 2 . The pH optimum for the reaction was between 7.0 and 7.5. PA phosphatase activity followed surface dilution kinetics with respect to PA ( K m = 5 mol%) using Triton X‐100‐PA mixed micelles. Lipin 1 enzyme activity was potently inhibited by N ‐ethylmaleimide with an IC 50 value of 50 μM. Within a conserved C‐terminal domain of lipin 1 is a DxDxT catalytic motif that is found in a superfamily of Mg 2+ ‐dependent phosphatase enzymes. Mutational analysis showed that lipin 1 proteins with aspartate‐to‐glutamate mutations (D678E and D680E) are catalytically inactive, confirming that the conserved aspartate residues are essential for Mg 2+ ‐dependent PA phosphatase activity. Supported by NIH grant GM 28140.