Crystal structure of a family <scp>II</scp> pyrophosphatase from <i>Thermodesulfobacterium commune</i> and factors enabling its high‐temperature adaptation

Inorganic pyrophosphatases (PPases) are crucial for energy metabolism and are classified into families with distinct metal ion requirements and structural features. Here, we report the expression, purification, and crystal structure of a thermostable family II PPase from the thermophile Thermodesulfobacterium commune (TcPPase). TcPPase, which is optimally activated by Co²⁺ and Mn²⁺, requires both the N- and C-terminal domains for full catalytic activity. Comparative structural analyses with orthologous enzymes from psychrophilic and mesophilic organisms suggest that the thermostability of TcPPase is attributable to enhanced hydrophobic interactions, increased proline content, dense hydrogen-bonding networks, and additional salt bridges. These findings reveal the molecular basis for the thermal adaptation of family II PPases, providing valuable insights for thermostable enzyme engineering for biotechnological applications.