Hill Samuel G, DeFeo Katherine, Offenbacher Adam R
Department of Chemistry, East Carolina University, Greenville, NC 27858, USA.
Biomolecules. 2025 Aug 11;15(8):1153. doi: 10.3390/biom15081153.
Lipoxygenases (LOXs) are a family of metalloenzymes that oxidize polyunsaturated fatty acids producing cell-signaling hydroperoxides. Fungal LOXs have drawn interest because of their roles in plant and animal pathogenesis. A new subfamily of annotated fungal LOXs has been predicted. One of its unique structural features is the presence of a cysteine amino acid encoded at the invariant leucine clamp. Herein, we isolate three representatives of this LOX subfamily from recombinant expressions in both yeast and bacterial cultures. Metal analysis indicates that the proteins accommodate a mononuclear manganese ion center, similar to other eukaryotic LOXs, but have nominal LOX activity. The functional consequence of the non-conservative mutation is further explored using a Leu-to-Cys (L546C) variant of soybean lipoxygenase, a model plant orthologue. While this L546C variant has comparable structural integrity and metal content to the native enzyme, the variant is associated with a 50-fold decrease in the first-order rate constant. The presence of cysteine at 546, compared to leucine, alanine, or serine, also results in a distinctive kinetic lag phase and product inhibition. The collective data highlight that Cys encoded at the Leu clamp is detrimental to LOX activity. Potential biological functions of these annotated fungal LOXs are discussed.
脂氧合酶（LOXs）是一类金属酶，可氧化多不饱和脂肪酸，产生细胞信号传导氢过氧化物。真菌脂氧合酶因其在植物和动物发病机制中的作用而受到关注。一个新的注释真菌脂氧合酶亚家族已被预测。其独特的结构特征之一是在不变的亮氨酸钳位编码的半胱氨酸氨基酸的存在。在此，我们从酵母和细菌培养物中的重组表达中分离出该脂氧合酶亚家族的三个代表。金属分析表明，这些蛋白质容纳一个单核锰离子中心，与其他真核脂氧合酶类似，但具有标称的脂氧合酶活性。使用大豆脂氧合酶（一种模型植物直系同源物）的亮氨酸到半胱氨酸（L546C）变体进一步探索了非保守突变的功能后果。虽然这种L546C变体与天然酶具有可比的结构完整性和金属含量，但该变体的一级速率常数降低了50倍。与亮氨酸、丙氨酸或丝氨酸相比，546位半胱氨酸的存在还导致明显的动力学滞后阶段和产物抑制。这些数据共同表明，亮氨酸钳位处编码的半胱氨酸对脂氧合酶活性有害。本文还讨论了这些注释真菌脂氧合酶的潜在生物学功能。
