Substrate-Binding Cavity Engineering of the Lipoxygenase from <i>Pseudomonas aeruginosa</i> to Produce 8<i>S</i>- and 11<i>S</i>-Hydroxyeicosatetraenoic Acids

Lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids. Notably, most microbial lipoxygenases including the lipoxygenase from Pseudomonas aeruginosa (Pa-LOX) catalyze oxygenation of linoleic acid and arachidonic acid into 13S-hydroperoxyoctadecenoic acid (13S-HPODE) and 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE), respectively. Therefore, this study has focused on modification of positional specificity or regioselectivity of Pa-LOX. The linoleic acid oxygenations and substrate-docking simulations suggested that the regioselectivity of Pa-LOX might depend on the geometry of the hydrocarbon tail-binding cavity. Therefore, the interior end of the substrate-binding cavity was enlarged to make C10 instead of C13 face the iron active site. Remarkably, the M434G mutation led to alteration of the oxygenation products from 15S-hydroxyeicosatetraenoic acid (15S-HPETE) to 11S-HPETE as the major product from arachidonic acid. On the other hand, the Y609G substitution allowed the formation of 8S-HPETE from arachidonic acid. 8S-HPETE was recovered after reduction by tris(2-carboxyethyl)phosphine hydrochloride with an isolated yield of 62% with a purity of 94% via Escherichia coli-based whole-cell biocatalysis, solvent extraction, and silica gel chromatography. This is the first report of the production of 11S-HPETE and 8S-HPETE from arachidonic acid at high conversions. Therefore, this study contributes to the preparation of biologically active oxylipins from renewable fatty acids in a sustainable way.