Flipped Regiospecificity in L434F Mutant of 8-Lipoxygenase

Lipoxygenases are non-heme iron containing enzymes that catalyze oxygenation of poly-unsaturated fatty acids in different animal and plant species with extremely high regio and stereospecificity. Nature employs 8-lipoxygenase to produce 8R-hydroperoxide product from the oxygenation of arachidonic acid. A single point L434F mutation of 8-lipoxygenase alters the regio- and stereo-specificity of the final products, with a product ratio of 66:34 for 8R- and 12S-hydroperoxides, respectively. A molecular level explanation of this flipped regiospecificity is presented in this work on the basis of extensive molecular dynamics simulations and kinetic network analysis of oxygen migration in the protein matrix. Phe434 is shown to exist in two conformations, the so-called open and closed conformations. In the closed conformation, the phenyl group of Phe434 shields the C8 site of substrate, thereby preventing accumulation of oxygen density at this site, which leads to a quenching of 8R product. On the other hand, both closed and open conformations of Phe434 allow oxygen density to accumulate at the pro-S face of C12 site of substrate, which enhances the propensity of 12S-hydroperoxides.