Fig. 1
From: Structural insights into the committed step of bacterial phospholipid biosynthesis
Characterization of aaPlsY in LCP. a The principle of the Pi-biosensor assay. The sticky LCP loaded with aaPlsY and its substrate 16:0-P is dispensed at the side of a microplate well and is repeatedly soaked to remove contaminant Pi. G3P, pre-treated enzymatically with a Pi-scavenger14, diffuses from the bathing solution to LCP, and initiates the reaction. The product Pi is then released from LCP to the bathing solution, triggering fluorescence increase of the Pi-biosensor14, which is monitored continuously using a plate reader. b Progress curves of the coupled assay omitting G3P (magenta), 16:0-P (cyan), enzyme (blue), and none of the above (black). The 2 min lag phase (indicated by an arrow) reflects the time required for equilibration of G3P between the bathing solution and LCP16; the linear phase is used to calculate activity. c Thin layer chromatography assay of aaPlsY activity. Lane 1, monoolein; lane 2, monoolein with 16:0-P (substrate); lane 3, monoolein with lysoPA (product); lane 4, monoolein with n-dodecyl-β-d-maltoside; lane 5, reaction without aaPlsY; lane 6, reaction with aaPlsY. The brightness and contrast of the whole image have been adjusted to enhance the substrate and product bands. d, e Michaelis–Menten and Lineweaver–Burk plot (insets) of kinetic measurements for the indicated substrates. f Dependence of activity rate on enzyme loading. In d–f, data represent the average ± SD from triplicate measurements
