Structure and mechanism of mammalian stearoyl-CoA desaturases

哺乳动物硬脂酰辅酶A去饱和酶 (SCD) 是氧化还原酶超家族的成员，具有 四个跨膜螺旋和一个由九个保守组氨酸残基组成的二铁中心。这个 超级家族拥有来自各个生命王国的 20,000 多名成员，但人们对其功能知之甚少 以及这些蛋白质的作用机制。该提案的总体目标是了解 SCD 的机制 原子分辨率。 SCD 驻留在内质网膜上并催化形成 饱和脂肪酰辅酶A上的双键。 SCD 活性降低的小鼠不会变得肥胖或糖尿病 当喂食高脂肪饮食时，说明 SCD 活性在能量稳态中的重要性。 SCD 由于对膜生物合成的更高需求，癌细胞中的表达水平上调，并且 抑制 SCD 活性可阻止癌细胞生长。这些结果导致了针对 SCD 的大力努力 肥胖、糖尿病和癌症的治疗，但由于缺乏 3 维数据，这些努力受到阻碍 SCD 的结构和催化机制的不完全理解。 目前尚不清楚 SCD 如何识别其底物并实现双键特异性 形成。全组氨酸配位的二铁中心不同于已知结构的其他二铁中心 其催化机制尚未得到研究。此外，持续的 SCD 活动需要两个 其他膜蛋白、细胞色素 b5 (b5) 和细胞色素 b5 还原酶 (b5R)，介导电子 从 NADH 转移到 Diiron 中心。然而，二铁中心如何通过动态相互作用进行氧化还原循环 b5和b5R仍然是个谜。 我们表达并纯化了功能性小鼠 SCD1，并将其晶体结构解析至 2.6 Å 分辨率。 我们还表达并纯化了全长b5和b5R，并在体外组装了酶促反应。在 此外，我们在b5R和b5之间以及b5和SCD1之间产生了稳定的二元复合物，并且我们 建立了测量结合配偶体之间电子转移的测定方法。这些初步结果 使我们能够提出以下三个具体目标： 目标 1：表征 SCD1 的结构和功能。 目标 2：了解 SCD1 二铁中心的氧化和还原途径。 目标 3：了解 b5 和 SCD1 之间以及 b5R 之间电子转移的结构基础 和b5。

Mammalian stearoyl-CoA desaturase (SCD) is a member of a super family of redox enzymes that have four transmembrane helices and a diiron center composed of nine conserved histidine residues. This superfamily has over 20,000 members from all kingdoms of life and yet very little is known about the function and mechanism of these proteins. The overall goal of the proposal is to understand the mechanism of SCD at the atomic resolution. SCD resides on endoplasmic reticulum membrane and catalyzes the formation of a double bond on saturated fatty acyl-CoAs. Mice with reduced SCD activity do not become obese or diabetic when fed with a high-fat diet, illustrating the significance of SCD activity in energy homeostasis. SCD expression level is upregulated in cancer cells because of a higher demand for membrane biosynthesis, and inhibition of SCD activity thwarts cancer cell growth. These results led to intense efforts on targeting SCDs for the treatment of obesity, diabetes, and cancer, but the efforts are hampered by a lack of 3-dimenional structures of SCD and incomplete understanding of the catalytic mechanism. It remains unclear how SCDs recognize their substrates and achieve specificity in double bond formation. The all-histidine coordinated diiron center is different from other diiron centers of known structures and its mechanism of catalysis has not been examined. In addition, sustained SCD activity requires two additional membrane proteins, cytochrome b5 (b5) and cytochrome b5 reductase (b5R), which mediate electron transfer from NADH to the diiron center. Yet how the diiron center is redox-cycled through dynamic interactions with b5 and b5R remains a mystery. We expressed and purified functional mouse SCD1 and solved its crystal structure to 2.6 Å resolution. We also expressed and purified full length b5 and b5R, and assembled the enzymatic reactions in vitro. In addition, we produced stable binary complexes between b5R and b5 and between b5 and SCD1, and we established assays to measure electron transfer between the binding partners. These preliminary results allowed us to propose the following three Specific Aims: Aim 1: To characterize the structure and function of SCD1. Aim 2: To understand the oxidative and reductive pathways of the SCD1 diiron center. Aim 3: To understand the structural basis of electron transfer between b5 and SCD1 and between b5R and b5.