Fig. 5: Rotameric Histidine switch and proposed catalytic mechanism of yCPT1.

a Structural organization and the cryo-EM density map of the rotameric His118 conformations, water molecule, Glu114, Asp121, and CDP-choline. CDP-choline, Glu114, Asp121, and His118 are depicted as sticks and water molecule is shown as a red ball. b Validation of the critical roles of His118 and Glu114 in the catalytic activity of yCPT1. Choline phosphotransferase activity of yCPT1 WT (blue circles), yCPT1 E114A (green triangles) and yCPT1 H118A (red squares) was assessed by measuring the amount of CMP released from CDP-choline at the indicated concentration in the presence of DAG 16:0-16:0 (100 μM). Data are presented as the mean ± SD from three independent measurements. Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test. c (1) In the apo-state, His118 mostly adopts an “out” conformation pointing away from the catalytic center. (2) Upon the binding of DAG, the equilibrium of His118 moves toward “in” conformation closer to the catalytic center. (3) The binding of CDP-choline further stabilizes the equilibrium of His118 to the “in” position (4) His118 at the “in” position deprotonates 3-hydroxyl of DAG to trigger nucleophilic substitution reaction toward the phosphate group of CDP-choline. (5) End-products PC and CMP are released from their binding sites and yCPT1 returns to the apo-state. Source data are provided as a Source Data file.