Combined computational and experimental studies reveal an altered regiospecific switch in 5-lipoxygenase targeted by...

5-Lipoxygenase (5-LOX) regulates leukotriene biosynthesis by oxygenating arachidonic acid (AA), and its products are key mediators of inflammatory signaling. 3-O-acetyl-11-keto-β-boswellic acid (AKBA), a boswellic acid derivative, shows allosteric inhibition against 5-LOX that shifts the enzyme's regiospecificity from 5-hydroperoxyeicosatetraenoic acid [5-H(p)ETE] to 12-hydroperoxyeicosatetraenoic acid [12-H(p)ETE]. In the current study, the molecular basis of AKBA-induced regiospecificity switching was investigated by integrating molecular docking and molecular dynamics simulations, with further validation by site-directed mutagenesis and cellular oxylipin analysis. AKBA binding promoted a reorientation of the aliphatic chain of AA within the active site, displacing the C7 carbon away from the catalytic iron while favouring a C10-proximal geometry associated with increased 12-H(p)ETE formation. Mutations at R101 and E108 weakened these AKBA-induced effects and diminished the shift toward 12-H(p)ETE formation. These findings demonstrate that AKBA rewires 5-LOX regiospecificity through coordinated substrate repositioning and allosteric control of domain-level dynamics, highlighting a mechanistic framework for allosteric regulation of lipoxygenase activity.