title: Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure.
abstract: Human platelet 12-lipoxygenase (hp-12LOX, 662 residues+iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a high-resolution X-ray structure is growing crystals, a process that requires the preparation of highly homogenous, reproducible and stable protein samples. To understand the properties of hp-12LOX, we have expressed and studied the behavior, function and low-resolution structure of the hp-12LOX His-tagged recombinant enzyme and its mutants in solution. We have found that it is a dimer easily converted into bigger aggregates, which are soluble/covalent-noncovalent/reversible. The heavier oligomers show a higher activity at pH 8, in contrast to dimers with lower activity showing two maxima at pH 7 and pH 8, indicating the existence of two different conformers. In the seven-point C-->S mutant, aggregation is diminished, activity has one broad peak at pH 8 and there is no change in specificity. Truncation of the N(t)-beta-barrel domain (PLAT, residues 1-116) reduces activity to approximately 20% of that shown by the whole enzyme, does not affect regio- or stereospecificity and lowers membrane binding by a factor of approximately 2. "NoPLAT" mutants show strong aggregation into oligomers containing six or more catalytic domains regardless of the status of the seven cysteine residues tested. Time-of-flight mass spectrometry suggests two arachidonic acid molecules bound to one molecule of enzyme. Small angle X-ray scattering studies (16 A resolution, chi approximately 1) suggest that two hp-12LOX monomers are joined by the catalytic domains, with the PLAT domains floating on the flexible linkers away from the main body of the dimer.
authors.affiliation: Urology Research Center, College of Medicine, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA.
authors.forename: Ansari M
authors.initials: AM
authors.lastname: Aleem
authors.forename: Jerzy
authors.initials: J
authors.lastname: Jankun
authors.forename: John D
authors.initials: JD
authors.lastname: Dignam
authors.forename: Matthias
authors.initials: M
authors.lastname: Walther
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kühn
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Ewa
authors.initials: E
authors.lastname: Skrzypczak-Jankun
mesh_terms: D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D019281:Dimerization; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D016718:Arachidonic Acid; D001092:Arachidonate 12-Lipoxygenase; C476558:ALOX12 protein, human