(A) Intrinsic ATPase activity of Fam20C, Fam20C D478A (DA) and Fam20A. Recombinant proteins were incubated with 0.5 mM ATP and 10 mM MnCl2 at 30°C for the indicated time. The amount of phosphate released during incubation was determined using malachite green reagent. (B) Thermal stability shift assay of MBP, Fam20A and Fam20C. Protein thermal stability was monitored by the fluorescence generated from binding of the dye SYPRO Orange to the hydrophobic region exposed upon protein denaturation. MBP purified the same way as Fam20A and Fam20C was used as a negative control. Reaction buffer contained 10 mM MnCl2. (C) Sequence alignment of Fam20A and Fam20C protein orthologs (Hs, Homo sapiens; Gg, Gallus gallus; Xt, Xenopus tropicalis; Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans). Catalytically important residues are highlighted and numbered according to human Fam20C. See Figure 2—figure supplement 1 for an extended alignment. (D) Fam20C active site from PDB:4kqb bound to ADP and Mn2+ ions. Conserved canonical kinase ion pair, ion interacting, and catalytic residues are highlighted (cyan sticks) and labeled according to human Fam20C. A Fam20-specific loop is colored in green and contributes a unique active site residue E306. (E) Intrinsic ATPase activities of Fam20A Q258E and Fam20C E306Q. Reactions were carried out for 1 hr. (F) Effect of Q258E mutation on Fam20A kinase activity. Recombinant OPN was used as the substrate.