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  • In vitro analysis of the Staphylococcus aureus lipoteichoic acid synthase enzyme using fluorescently labeled lipids.

In vitro analysis of the Staphylococcus aureus lipoteichoic acid synthase enzyme using fluorescently labeled lipids.

Journal of bacteriology (2010-08-17)
Maria Karatsa-Dodgson, Mirka E Wörmann, Angelika Gründling
ABSTRACT

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. The key enzyme responsible for polyglycerolphosphate lipoteichoic acid synthesis in the Gram-positive pathogen Staphylococcus aureus is the membrane-embedded lipoteichoic acid synthase enzyme, LtaS. It is presumed that LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. Here we describe an in vitro assay for this new class of enzyme using PG with a fluorescently labeled fatty acid chain (NBD-PG) as the substrate and the recombinant soluble C-terminal enzymatic domain of LtaS (eLtaS). Thin-layer chromatography and mass spectrometry analysis of the lipid reaction products revealed that eLtaS is sufficient to cleave the glycerolphosphate head group from NBD-PG, resulting in the formation of NBD-diacylglycerol. An excess of soluble glycerolphosphate could not compete with the hydrolysis of the fluorescently labeled PG lipid substrate, in contrast to the addition of unlabeled PG. This indicates that the enzyme recognizes and binds other parts of the lipid substrate, besides the glycerolphosphate head group. Furthermore, eLtaS activity was Mn(2+) ion dependent; Mg(2+) and Ca(2+) supported only weak enzyme activity. Addition of Zn(2+) or EDTA inhibited enzyme activity even in the presence of Mn(2+). The pH optimum of the enzyme was 6.5, characteristic for an enzyme that functions extracellularly. Lastly, we show that the in vitro assay can be used to study the enzyme activities of other members of the lipoteichoic acid synthase enzyme family.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-α-Phosphatidyl-DL-glycerol sodium salt from egg yolk lecithin, ≥99% (TLC), lyophilized powder
Avanti
16:0-06:0 NBD PE, Avanti Research - A Croda Brand 810153C
Avanti
16:0-06:0 NBD PE, Avanti Research - A Croda Brand 810153P, powder
Avanti
16:0-06:0 NBD PS, Avanti Research - A Croda Brand 810192P, powder
Avanti
16:0-06:0 NBD PS, Avanti Research - A Croda Brand 810192C
Avanti
14:0-06:0 NBD PG, Avanti Research - A Croda Brand 810161P, powder
Avanti
16:0-12:0 NBD PE, Avanti Research - A Croda Brand 810154P, powder
Avanti
16:0-06:0 NBD PC, Avanti Research - A Croda Brand 810130C
Avanti
16:0-06:0 NBD PC, Avanti Research - A Croda Brand 810130P, powder
Avanti
16:0-06:0 NBD PG, Avanti Research - A Croda Brand 810163P, powder
Avanti
Egg PG, Avanti Research - A Croda Brand
Avanti
Egg PG, Avanti Research - A Croda Brand