- Developing a High-Throughput Assay for the Integral Membrane Glycerol 3-Phosphate Acyltransferase.
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 30โmin. PlsY showed Michaelis-Menten kinetics behavior in micelles with a V max of 57.5โฮผmol min-1 mg-1, and K m of 1.14โmM G3P and 6.2โฮผM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC50 of 19โฮผM. 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.