UC Santa Cruz

Human 15-lipoxygenases (LOX) are critical enzymes in the inflammatory process, producing various pro-resolution molecules, such as lipoxins and resolvins, but the exact role each of the two 15-LOXs in these biosynthetic pathways remains elusive. Previously, it was observed that h15-LOX-1 reacted with 5S-HETE in a non-canonical manner, producing primarily the 5S,12S-diHETE product. To determine the active site constraints of h15-LOX-1 in achieving this reactivity, amino acids involved in the fatty acid binding were investigated. It was observed that R402L did not have a large effect on 5S-HETE catalysis, but F414 appeared to π-π stack with 5S-HETE, as seen with AA binding, indicating an aromatic interaction between a double bond of 5S-HETE and F414. Decreasing the size of F352 and I417 shifted oxygenation of 5S-HETE to C12, while increasing the size of these residues reversed the positional specificity of 5S-HETE to C15. Mutants at these locations demonstrated a similar effect with 7S-HDHA as the substrate, indicating that the depth of the active site regulates product specificity for both substrates. Together, these data indicate that of the three regions proposed to control positional specificity, π-π stacking and active site cavity depth are the primary determinants of positional specificity with 5S-HETE and h15-LOX-1. Finally, the altered reactivity of h15-LOX-1 was also observed with 5S-HEPE, producing 5S,12S-diHEPE instead of 5S,15S-diHEPE (aka resolvin E₄ (RvE₄). However, h15-LOX-2 efficiently produces 5S,15S-diHEPE from 5S-HEPE. This result is important with respect to the biosynthesis of the RvE₄ since it obscures which LOX isozyme is involved in its biosynthesis. Future work detailing the expression levels of the lipoxygenase isoforms in immune cells and selective inhibition during the inflammatory response will be required for a comprehensive understanding of RvE₄ biosynthesis.