title: Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system.
authors: Institut für Biochemie, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors: Swathi
authors: S
authors: Banthiya
authors: Institut für Medizinische Physik und Biophysik, Group Protein X-ray Crystallography and Signal Transduction, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors: Jacqueline
authors: J
authors: Kalms
authors: Institut für Medizinische Physik und Biophysik, Group Protein X-ray Crystallography and Signal Transduction, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors: Etienne
authors: E
authors: Galemou Yoga
authors: Institut für Biochemie, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors: Igor
authors: I
authors: Ivanov
authors: Institut de Biologia Molecular de Barcelona (IBMB-CSIC), Parc Científic de Barcelona, 08028 Barcelona, Spain; XALOC beamline, ALBA synchrotron (CELLS), 08290 Cerdanyola del Vallès, Spain.
authors: Xavi
authors: X
authors: Carpena
authors: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors: Mats
authors: M
authors: Hamberg
authors: Institut für Biochemie, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. Electronic address: hartmut.kuehn@charite.de.
authors: Hartmut
authors: H
authors: Kuhn
authors: Institut für Medizinische Physik und Biophysik, Group Protein X-ray Crystallography and Signal Transduction, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. Electronic address: patrick.scheerer@charite.de.
authors: Patrick
authors: P
authors: Scheerer
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004795:Enzyme Stability; D005227:Fatty Acids / Q000378:metabolism; D007700:Kinetics; D015289:Leukotrienes / Q000378:metabolism; D008024:Ligands; D019787:Linoleic Acid / Q000737:chemistry / Q000378:metabolism; D044045:Lipoxins / Q000096:biosynthesis; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005227:Fatty Acids; D015289:Leukotrienes; D008024:Ligands; D044045:Lipoxins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D019787:Linoleic Acid; D008084:Lipoxygenase
keywords: Bacteria; Biomembranes; Eicosanoids; Infection; Inflammation; Protein X-ray crystallography; Protein structure