Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure

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 similar to 20% of that shown by the whole enzyme, does not affect regio-or stereospecificity and lowers membrane binding by a factor of similar to 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(similar to 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. (C) 2007 Elsevier Ltd. All rights reserved.