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Merck

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

Assay and drug development technologies (2019-08-14)
Yannan Tang, Dianfan Li
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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 30โ€‰min. PlsY showed Michaelis-Menten kinetics behavior in micelles with a V max of 57.5โ€‰ฮผmol min-1 mg-1, and K m of 1.14โ€‰mM G3P and 6.2โ€‰ฮผM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC50 of 19โ€‰ฮผM. 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.

MATERIALS
Product Number
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Sigma-Aldrich
2,2-Dimethyl-1,3-propanediol, 99%
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Maltose solution, BioReagent, ~20% in H2O
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Glycerol phosphate disodium salt hydrate, isomeric mixture
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