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.

中文翻译：

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铜绿假单胞菌脂质氧合酶的底物结合腔工程产生 8S- 和 11S-羟基二十碳四烯酸


脂氧合酶催化多不饱和脂肪酸的双氧合。值得注意的是，大多数微生物脂氧合酶，包括来自铜绿假单胞菌的脂氧合酶 （Pa-LOX） 催化亚油酸和花生四烯酸分别氧化成 13个 S-氢过氧基十八碳烯酸 （13 S-HPODE） 和 15个 S-氢过氧二十碳四烯酸 （15S-HPETE）。因此，本研究的重点是改变 Pa-LOX 的位置特异性或区域选择性。亚油酸氧合和底物对接模拟表明，Pa-LOX 的区域选择性可能取决于烃尾结合腔的几何形状。因此，扩大了底物结合腔的内端，使 C10 而不是 C13 面向铁活性位点。值得注意的是，M434G 突变导致氧合产物从 15S-羟基二十碳四烯酸 （15 S-HPETE） 改变为 11S-HPETE 作为花生四烯酸的主要产物。另一方面，Y609G 取代允许从花生四烯酸形成 8 S-HPETE。8 S-HPETE 经盐酸三（2-羧乙基）膦还原后，通过基于大肠杆菌的全细胞生物催化、溶剂萃取和硅胶色谱法回收，分离收率为 62%，纯度为 94%。这是从花生四烯酸以高转化率生产 11 S-HPETE 和 8S-HPETE 的首次报告。因此，本研究有助于以可持续的方式从可再生脂肪酸制备具有生物活性的脂氧化物。