Mutations of Triad Determinants Changes the Substrate Alignment at the Catalytic Center of Human ALOX5

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 wildtype 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 wildtype ALOX5 converted arachidonic acid mainly to 5(S)-HpETE. In contrast, 15(S)- and 8(S)-H(p)ETE were formed by the mutant enzyme. In addition to arachidonic acid, wildtype ALOX5 accepted EPA as substrate but C18 fatty acids were not oxygenated. The quadruple mutant also accepted linoleic acid, alpha- and gamma-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 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.