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Updated: Dec 20, 2025
Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening
Published on: May 13, 2020
Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to
Karine Nozeret1, Aurélia Pernin1, Nienke Buddelmeijer2
1Institut Pasteur, Unité Biologie et génétique de la paroi bactérienne, Paris, France ; CNRS, UMR 2001 « Microbiologie intégrative et Moléculaire », Paris, France ; INSERM, Équipe Avenir, Paris, France.
A new assay detects the activity of apolipoprotein N-acyltransferase (Lnt), an enzyme crucial for bacterial lipoprotein modification. This high-throughput screening method aids in developing novel antibacterial agents targeting essential bacterial pathways.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Proteobacteria utilize a three-enzyme pathway for post-translational modification of lipoproteins, involving sequential fatty acid acylation.
- Apolipoprotein N-acyltransferase (Lnt) catalyzes the final acylation step, which is essential for most γ-proteobacteria.
- This pathway represents a promising target for novel antibacterial agent development.
Purpose of the Study:
- To develop a sensitive, high-throughput screening (HTS)-compatible assay for apolipoprotein N-acyltransferase (Lnt) activity.
- To facilitate the discovery of small inhibitory molecules targeting bacterial lipoprotein modification.
Main Methods:
- Development of an in vitro assay for membrane-embedded Lnt enzyme and its substrates.
- Purification of active Lnt in detergent and preparation of alkyne-phospholipid and diacylglyceryl peptide substrates.
- A fluorometric assay utilizing click chemistry to detect the fluorescently labeled triacylated peptide product in a multiwell format.
Main Results:
- A novel fluorometric assay for Lnt activity was established, suitable for HTS.
- The assay directly detects the reaction product, avoiding coupled enzymatic reactions.
- The method is adaptable for other acyltransferases utilizing fatty acid-containing substrates.
Conclusions:
- The developed assay provides a robust platform for screening Lnt inhibitors.
- This assay can accelerate the identification of new antibacterial compounds targeting essential bacterial enzymes.
- The assay's adaptability broadens its utility for studying related acyltransferase enzymes.
