2DG6P phosphatase assays were performed in 250 μl of reaction containing 1.5 mM 2DG6P (#17149, Cayman Chemical, Ann Arbor, Michigan, USA) in 50 mM Hepes (pH 6.7), 10 mM MgCl2, and 10% glycerol and 30 μg of recombinant HDHD1 or HDHD1-DDAA. Samples were incubated at 37°C for various times (0, 5, 10, and 15 min) and the reaction was stopped by adding 150 μl of EDTA (0.5 M). Then, the 2DG generated was assayed by adding 500 μl of glucose assay reagent (GAGO20, Sigma-Aldrich) and further incubating at 37°C for 30 min. The reaction was stopped by adding 500 μl of H2SO4 (12N), and the absorbance of the reaction was measured at 540 nm. A slope (A540 over time) was calculated to assess enzyme activity and to make sure that the reaction was in the linear range. The measurements were repeated three times.

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