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

Abstract

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 Vmax of 57.5 μmol min-1 mg-1, and Kmof 1.14 mM G3P and 6.2 μM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC50 of 19 μM. The results principally demonstrated the feasibility of using the assay for high-throughput screening, and the protocol provided an encouraging starting point for further optimization and validation of the assay for automated platforms.