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

Area of Science:

• Biochemistry
• Microbiology
• Drug Discovery

Background:

• Phospholipid biosynthesis is essential for bacterial survival, with PlsY catalyzing the initial acylation of glycerol 3-phosphate (G3P).
• PlsY is a validated antibiotic target in Gram-positive bacteria, including pathogens, due to its essential role.
• Previous enzymatic assays using lipid cubic phases (LCP) faced limitations in high-throughput applications due to viscosity.

Purpose of the Study:

• To develop a high-throughput compatible enzymatic assay for PlsY.
• To characterize PlsY kinetics and inhibition in a micellar environment.

Main Methods:

• Adapted a previously developed continuous phosphate-monitoring assay.
• Replaced the LCP environment with detergent micelles to host PlsY.
• Utilized standard multi-channel pipets for high-throughput screening.

Main Results:

• The assay demonstrated linear reaction velocity for up to 30 minutes with optimal enzyme loading.
• PlsY exhibited Michaelis-Menten kinetics in micelles: Vmax = 57.5 μmol min⁻¹ mg⁻¹, K_M (G3P) = 1.14 mM, K_M (acyl phosphate) = 6.2 μM.
• The inhibitory product lysophosphatidic acid was characterized.

Conclusions:

• The micelle-based assay is suitable for high-throughput screening of PlsY inhibitors.
• This assay facilitates the discovery of novel antibiotics targeting bacterial phospholipid biosynthesis.