Abstract
Lipoprotein modification is an essential process in Gram-negative bacteria. The action of three integral membrane proteins that catalyze the transfer of fatty acids derived from membrane phospholipids or cleave the signal peptide of the lipoprotein substrate result in the formation of mature triacylated proteins. Inactivation of the enzymes leads to mis-localization of immature lipoproteins and consequently cell death. Biochemical studies and the development of in vitro assays are challenging due to the fact that the enzymes and substrates are all membrane-embedded proteins difficult to overproduce and purify. Here we describe a sensitive fluorescence-based assay to monitor bacterial apolipoprotein N-acyltransferase activity.
Highlights
IntroductionIn Gram-negative (proteo-) bacteria, three integral membrane proteins catalyze the formation of triacylated proteins; prolipoprotein diacylglyceryl transferase (Lgt), signal peptidase II (Lsp) and apolipoprotein N-acyltransferase (Lnt)[1] (Fig. 1A)
The lipoprotein modification pathway is essential for viability in bacteria
FSL-1 is a small decapeptide that contains a diacylglyceryl group at the N-terminal cysteine residue to mimic the natural apolipoprotein substrate that is extensively used as immune stimulating molecule through Toll-like receptor 2 (TLR2) signaling pathways[12]
Summary
In Gram-negative (proteo-) bacteria, three integral membrane proteins catalyze the formation of triacylated proteins; prolipoprotein diacylglyceryl transferase (Lgt), signal peptidase II (Lsp) and apolipoprotein N-acyltransferase (Lnt)[1] (Fig. 1A). Lgt and Lnt use membrane phospholipids as acyl donor and prolipoprotein and apolipoprotein (S-diacylglyceryl protein) as protein substrate in the reaction, respectively. A second assay includes coupled enzymatic reactions that has been proposed to screen for Lgt inhibitors in vitro[10] This approach is based on the conversion of glycerol-1-phosphate, the by-product of the Lgt reaction, into glycerol and phosphate by the addition of alkaline phosphatase and subsequent formation of dihydroxyacetone and NADH from glycerol by glycerol-dehydrogenase. Product formation was analyzed by in-gel fluorescence and fluorescence spectroscopy in 96-well plate format This sensitive assay allows detailed characterization of the molecular mechanism of acyltransferases and the development of a high-throughput-screen (HTS) set-up for screening of specific inhibitors
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