Fig. 4

Interactions between aaPlsY and its substrates 16 : 0-P and G3P, and structure-based mutagenesis. a Interactions between the acylP head group and the enzyme. b The involvement of two α-helices in acylP binding. The aggregate effect of individual backbone microdipoles aligned along the α-helices axis (black arrows) causes a dipole moment with its positive pole at the N terminus and its negative pole at the C terminus. The resultant partial positive charges (δ⁺) at the N termini of the α2 (n = 10) and the TMH4 (n = 13) neutralize the phosphate charges of 16 : 0-P. c The hydrophobic groove that hosts the acyl chain of 16 : 0-P. Residues within 4.0 Å of the palmitoyl chain are shown as green sticks. d The G3P binding pocket. Dashed lines in a and d indicate distances within 3.2 Å. When appropriate, the dashed lines are colored differently for better visualization. e The activity of the wild-type and mutant enzymes. The wild-type activity (34.55 ± 3.14 μmol min⁻¹ mg⁻¹, mean ± SD) was obtained from six independent experiments, and the mean value was set to 100%. Purified mutants were assayed under eleven different G3P concentrations without technical replicates. G3P concentrations typically span three orders of magnitude with the high concentrations saturating the system. The V _max and SE from the Michaelis–Menten fitting are reported. Residues involved in binding of the two substrates are indicated appropriately