A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis

Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding prot...

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Bibliographic Details
Published in:The Journal of biological chemistry 2018-02, Vol.293 (5), p.1493-1503
Main Authors: Choi, Jae-Yeon, Surovtseva, Yulia V., Van Sickle, Sam M., Kumpf, Jan, Bunz, Uwe H.F., Ben Mamoun, Choukri, Voelker, Dennis R.
Format: Article
Language:English
Subjects:
Carboxy-Lyases - chemistry
Carboxy-Lyases - genetics
Fluorescence
inhibitor
inhibitor screening
Maltose-Binding Proteins - chemistry
Maltose-Binding Proteins - genetics
membrane biogenesis
Methods and Resources
Phosphatidylethanolamines - analysis
Phosphatidylethanolamines - chemistry
phosphatidylserine decarboxylase
phospholipid
Plasmodium knowlesi - enzymology
Plasmodium knowlesi - genetics
Protozoan Proteins - chemistry
Protozoan Proteins - genetics
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
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Summary:Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD (MBP-His6-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λex = 403 nm, λem = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.RA117.000525