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For the specificity of ALOX15 orthologs of different mammals, the geometry of the amino acids Phe353, Ile418, Met419, and Ile593 ("triad determinants") is important, and mutagenesis of these residues altered the reaction specificity of these enzymes. Here we expressed wild-type human ALOX5 and its F359W/A424I/N425M/A603I mutant in Sf9 insect cells and characterized the catalytic differences of the two enzyme variants. We found that wild-type ALOX5 converted arachidonic acid mainly to 5( S )-hydroperoxyeicosatetraenoic acid (HpETE). In contrast, 15( S )- and 8( S )-H(p)ETE were formed by the mutant enzyme. In addition to arachidonic acid, wild-type ALOX5 accepted eicosapentaenoic acid (EPA) as substrate, but C18 fatty acids were not oxygenated. The quadruple mutant also accepted linoleic acid and α- and γ-linolenic acid as substrate. Structural analysis of the oxygenation products and kinetic studies with stereospecifically labeled 11( S )- and 11( R )-deutero-linoleic acid suggested alternative ways of substrate orientation at the active site. In silico docking studies, molecular dynamics simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations confirmed this hypothesis. These data indicate that "triad determinant" mutagenesis alters the catalytic properties of ALOX5 abolishing its leukotriene synthase activity but improving its biosynthetic capacity for pro-resolving lipoxins.

The regulatory effect of two oxyderivatives of unsaturated fatty acids (oxylipins), 18-hydroxy-(9Z,12Z)-octadecadienoic acid (18-HODE) and 18-(9Z,12Z,15Z)-octadecatrienoic acid (18-HOTrE), on the sexual and asexual sporulation of wild-type Neurospora crassa strains and wc-1 and wc-1 mutants was studied. In the wild-type strain, 18-HODE, unlike 18-HOTrE, stimulated protoperithecia formation in the dark and in the light. In the same strain, the studied oxylipins influenced conidiagenesis only under illumination. 18-HODE stimulated and 18-HOTrE inhibited the conidia formation. Oxylipins had no effect on protoperithecia formation in photoreceptor complex mutants, which apparently indicated its involvement in signal transmission in N. crassa. The stimulating action of the studied oxylipins on conidiagenesis in wc-1 and the lack of action in wc-2 may indicate alternative signaling pathways of oxylipins in this process.