Identification of a highly conserved hypothetical protein TON_0340 as a probable manganese-dependent phosphatase

A hypothetical protein TON_0340 of a Thermococcus species is a protein conserved in a variety of organisms including human. Herein, we present four different crystal structures of TON-0340, leading to the identification of an active-site cavity harboring a metal-binding site composed of six invariant aspartate and glutamate residues that coordinate one to three metal ions. Biochemical and mutational analyses involving many phosphorous compounds show that TON-0340 is a Mn²⁺-dependent phosphatase. Mg²⁺ binds to TON-0340 less tightly and activates the phosphatase activity less efficiently than Mn2+. Whereas Ca²⁺ and Zn²⁺ are able to bind to the protein, they are unable to activate its enzymatic activity. Since the active-site cavity is small and largely composed of nearly invariant stretches of 11 or 13 amino acids, the physiological substrates of TON-0340 and its homologues are likely to be a small and the same molecule. The Mn²⁺-bound TON-0340 structure provides a canonical model for the ubiquitously present TON-0340 homologues and lays a strong foundation for the elucidation of their substrate and biological function.