Structural comparisons of phosphoenolpyruvate carboxykinases reveal the evolutionary trajectories of these phosphodiester energy conversion enzymes.

Inorganic pyrophosphate (PP) consists of two phosphate molecules and can act as an energy and phosphate donor in cellular reactions, similar to ATP. Several kinases use PP as a substrate, and these kinases have recently been suggested to have evolved from ATP-dependent functional homologs, which have significant amino acid sequence similarity to PP-utilizing enzymes. In contrast, phosphoenolpyruvate carboxykinase (PEPCK) can be divided into three types according to the phosphate donor (ATP, GTP, or PPi), and the amino acid sequence similarity of these PEPCKs is too low to confirm that they share a common ancestor. Here we solved the crystal structure of a PP-PEPCK homolog from the bacterium at 2.6 Å resolution and compared it with previously reported structures from ATP- and GTP-specific PEPCKs to assess the degrees of similarities and divergences among these PEPCKs. These comparisons revealed that they share a tertiary structure with significant value and that amino acid residues directly contributing to substrate recognition, except for those that recognize purine moieties, are conserved. Furthermore, the order of secondary structural elements between PP-, ATP-, and GTP-specific PEPCKs was strictly conserved. The structure-based comparisons of the three PEPCK types provide key insights into the structural basis of PP specificity and suggest that all of these PEPCKs are derived from a common ancestor.