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Arch Biochem Biophys. 2015 Oct 15;584:116-24. doi: 10.1016/j.abb.2015.09.003. Epub 2015 Sep 8.

Secreted lipoxygenase from Pseudomonas aeruginosa exhibits biomembrane oxygenase activity and induces hemolysis in human red blood cells.

Author information

1
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany.
2
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany; Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, 832 32 Bratislava, Slovakia.
3
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany. Electronic address: hartmut.kuehn@charite.de.

Abstract

Pseudomonas aeruginosa (PA) expresses a secreted lipoxygenase (LOX), which oxygenates free arachidonic acid predominantly to 15S-H(p)ETE. The enzyme is capable of binding phospholipids at its active site and physically interacts with model membranes. However, its membrane oxygenase activity has not been quantified. To address this question, we overexpressed PA-LOX as intracellular his-tag fusion protein in Escherichia coli, purified it to electrophoretic homogeneity and compared its biomembrane oxygenase activity with that of rabbit ALOX15. We found that both enzymes were capable of oxygenating mitochondrial membranes to specific oxygenation products and 13S-H(p)ODE and 15S-H(p)ETE esterified to phosphatidylcholine and phosphatidylethanolamine were identified as major oxygenation products. When normalized to similar linoleic acid oxygenase activity, the rabbit enzyme exhibited a much more effective mitochondrial membrane oxygenase activity. In contrast, during long-term incubations (24 h) with red blood cells PA-LOX induced significant (50%) hemolysis whereas rabbit ALOX15 was more or less ineffective. These data indicate the principle capability of PA-LOX of oxygenating membrane bound phospholipids which is likely to alter the barrier function of the biomembranes. Although the membrane oxygenase activity was lower than the fatty acid oxygenase activity of PA-LOX red blood cell membrane oxygenation might be of biological relevance for P. aeruginosa septicemia.

KEYWORDS:

Bacterial infections; Biomembranes; Eicosanoids; Oxidative stress

PMID:
26361973
DOI:
10.1016/j.abb.2015.09.003
[Indexed for MEDLINE]

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