A Gly-to-Ala Substitution Confers Choline Phosphotransferase Activity on the CDP-Ethanolamine-Specific Mammalian EPT1.

Choline phosphotransferase (CPT) and ethanolamine phosphotransferase (EPT) catalyze the synthesis of choline and ethanolamine phospholipids using CDP-choline or CDP-ethanolamine as substrates, respectively. Although both enzymes share the CDP-alcohol phosphotransferase motif at their active sites, the structural determinants of their substrate specificities remain unclear. Previously, we resolved the structure of the CDP-choline-specific yeast CPT1 and identified a Trp residue that interacts with the choline moiety of CDP-choline. The spatial positioning of this Trp residue was influenced by a neighboring Ala residue. Replacing this Ala with Gly conserved in EPT caused the Trp to shift inward, thereby conferring specificity for CDP-ethanolamine. Here we explored the reciprocal scenario in the CDP-ethanolamine-specific mammalian EPT1. A Gly105-to-Ala mutant of mouse EPT1 was generated, and structural prediction revealed an outward shift of Trp37 residue. Enzymatic assays and metabolic labeling in HEK293 cells demonstrated that the G105A mutant acquired CDP-choline specificity, which is absent in the wild-type enzyme. These results demonstrate that the positioning of Trp37, influenced by the adjacent Ala/Gly residue, is one of the important determinants of substrate specificity, supporting a conserved structural mechanism across yeast and mammalian systems.