Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa.

Lipoxygenases (LOXs), which are essential in eukaryotes, have no confirmed function in prokaryotes that are devoid of polyunsaturated fatty acids. The structure of a secretable LOX from Pseudomonas aeruginosa (Pa_LOX), the first available from a prokaryote, presents significant differences with respect to eukaryotic LOXs, including a cluster of helices acting as a lid to the active center. The mobility of the lid and the structural variability of the N-terminal region of Pa_LOX was confirmed by comparing 2 crystal forms. The binding pocket contains a phosphatidylethanolamine phospholipid with branches of 18 (sn-1) and 14/16 (sn-2) carbon atoms in length. Carbon atoms from the sn-1 chain approach the catalytic iron in a manner that sheds light on how the enzymatic reaction might proceed. The findings in these studies suggest that Pa_LOX has the capacity to extract and modify unsaturated phospholipids from eukaryotic membranes, allowing this LOX to play a role in the interaction of P. aeruginosa with host cells.

High conformational stability of secreted eukaryotic catalase-peroxidases: answers from first crystal structure and unfolding studies.

Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal (+)Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.