Developing a High-Throughput Assay for the Integral Membrane Glycerol 3-Phosphate Acyltransferase

Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acyltransferase that catalyzes this essential step, making it a potential target for the development of antibiotics. A convenient enzymatic assay should facilitate such drug discovery activities. Previously, we developed a continuous assay by monitoring phosphate, one of the enzymatic product, using a fluorescently labeled phosphate binding protein in a bilayer environment called lipid cubic phase (LCP). However, some intrinsic characteristics of LCP, such as high viscosity, make the assay incompatible with common high-throughput liquid-handling platforms. Here, we adapted the assay by hosting PlsY in detergent micelles, enabling us to conduct the assay using standard multi-channel pipets in a high-throughput manner. With optimal enzyme loading, the reaction velocity was linear up to 30 min. PlsY showed Michaelis-Menten kinetics behavior in micelles with a V of 57.5 μmol min^-1 mg^-1, and Kof 1.14 mM G3P and 6.2 μM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC