Omega-3 versus Omega-6 fatty acid availability is controlled by hydrophobic site geometries of phospholipase A2s

Human phospholipase A2s (PLA2) constitute a superfamily of enzymes that hydrolyze the sn-2 acyl-chain of glycerophospholipids, producing lysophos-pholipids and free fatty acids. Each PLA2 enzyme type contributes to specific biological functions based on its expression, subcellular localization, and substrate specificity. Among the PLA2 superfamily, the cytosolic cPLA2 enzymes, calcium-independent iPLA2 enzymes, and secreted sPLA2 enzymes are implicated in many diseases, but a central issue is the preference for double-bond positions in polyunsaturated fatty acids (PUFAs) occupying the sn-2 position of membrane phospholipids. We demonstrate that each PLA2 has a unique preference between the specific omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexae-noic acid (DHA) and the omega-6 arachidonic acid (AA), which are the precursors of most proinflammatory and anti-inflammatory or resolving eicosanoids and related oxylipins. Surprisingly, we discovered that human cPLA2 selectively prefers AA, whereas iPLA2 prefers EPA, and sPLA2 prefers DHA as substrate. We deter-mined the optimal binding of each phospholipid sub-strate in the active site of each PLA2 to explain these specificities. To investigate this, we utilized recently developed lipidomics-based LC-MS/MS and GC/MS assays to determine the sn-2 acyl chain specificity in mixtures of phospholipids. We performed mu s timescale molecular dynamics (MD) simulations to reveal unique active site properties, especially how the precise hy-drophobic cavity accommodation of the sn-2 acyl chain contributes to the stability of substrate binding and the specificity of each PLA2 for AA, EPA, or DHA. This study provides the first comprehensive picture of the unique substrate selectivity of each PLA2 for omega-3 and omega-6 fatty acids.