Abstract
Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties.
Highlights
Arachidonic acid lipoxygenases (ALOXs) isoforms form a family of lipid peroxidising enzymes, which metabolize free and esterified polyunsaturated fatty acids to bioactive hormones commonly named eicosanoids [1,2,3]
The molecular basis for the allosteric properties of ALOX-isoforms remains unclear but two alternative concepts have been suggested: (i) Allosteric ALOX-isoforms involve, in addition to the substrate-binding pocket, a binding site for allosteric regulators. Ligand binding at this hypothetic motif alters the structure of the substrate-binding pocket and the kinetics of substrate oxygenation [11,12]. (ii) Allosteric ALOX isoforms may function as dimeric proteins and one monomer within these dimers acts as allosteric regulator [13]
In previous studies we reported that tight association of the PLAT domain and the catalytic subunit of rabbit ALOX15 may be important for stability and integrity of α2 helix [20]
Summary
Arachidonic acid lipoxygenases (ALOXs) isoforms form a family of lipid peroxidising enzymes, which metabolize free and esterified polyunsaturated fatty acids to bioactive hormones commonly named eicosanoids [1,2,3]. The molecular basis for the allosteric properties of ALOX-isoforms remains unclear but two alternative concepts have been suggested: (i) Allosteric ALOX-isoforms involve, in addition to the substrate-binding pocket, a binding site for allosteric regulators Ligand binding at this hypothetic motif alters the structure of the substrate-binding pocket and the kinetics of substrate oxygenation [11,12]. (ii) Allosteric ALOX isoforms may function as dimeric proteins and one monomer within these dimers acts as allosteric regulator [13] In this scenario, the substrate binding pocket of the regulatory monomer constitutes the binding site for the allosteric ligand, whereas the substrate fatty acid is bound in the pocket of catalytic monomer
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