Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
FASEB J. 2012 Aug;26(8):3222-9. doi: 10.1096/fj.12-205286. Epub 2012 Apr 18.
The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca(2+)-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A(4) from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.
5-脂氧合酶（5-LOX）是一种酶，它启动促炎白三烯脂类介质的生物合成，与 15-脂氧合酶一起，也是合成抗炎脂氧素所必需的。5-LOX 的催化活性通过多种机制进行调节，包括 Ca2+靶向的膜结合和特定丝氨酸残基的磷酸化。为了研究 S663 磷酸化的后果，我们将该残基突变为磷酸化模拟物 Asp，提供了一种适合催化和结构研究的均一制剂。S663D 酶表现出强大的 15-LOX 活性，如分光光度法和 HPLC 分析所确定的，仅残留痕量的 5-LOX 活性；也检测到从花生四烯酸合成抗炎脂氧素 A（4）。在无和存在花生四烯酸的情况下（在先前报道的稳定 5-LOX 的背景下），S663D 突变体的晶体结构揭示了定义活性位点的螺旋的大量重塑，使得曾经完全包裹的催化机制变得可溶剂化。我们的结果表明，5-LOX 在 S663 处的磷酸化不仅可以下调白三烯的合成，还可以刺激不表达 15-LOX 的炎症细胞中脂氧素的产生，从而将脂质介质生物合成重新定向为产生促炎的抗炎介质。
