Structure and mechanism of mammalian stearoyl-CoA desaturases

哺乳动物硬脂酰辅酶A脱饱和酶(SCD)是氧化还原酶超家族中的一员，具有 四个跨膜螺旋和一个由九个保守的组氨酸残基组成的双铁中心。这 超级家族有来自所有生命王国的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。这项建议的整体目标是在 原子分辨率。SCD驻留在内质网膜上，催化a 饱和脂肪酰基-COAS上的双键。SCD活性降低的小鼠不会肥胖或糖尿病 当饲喂高脂饲料时，说明了SCD活性在能量平衡中的意义。SCD 由于对膜生物合成的更高要求，癌细胞中的表达水平上调，并且 抑制SCD活性会阻碍癌细胞的生长。这些结果导致了针对SCD的密集努力 肥胖症、糖尿病和癌症的治疗，但由于缺乏三维模型，这些努力受到阻碍 SCD的结构和对催化机理的不完全理解。 目前尚不清楚SCD如何识别它们的底物并在双键中实现特异性。 队形。全组氨酸配位的双铁中心不同于其他已知结构的双铁中心 其催化机理尚未得到研究。此外，持续的SCD活动需要两个 额外的膜蛋白，细胞色素b5(B5)和细胞色素b5还原酶(B5R)，它们介导电子 从NADH转到双铁中心。然而，双铁中心是如何通过动态相互作用进行氧化还原循环的 B5和b5R仍然是一个谜。 我们表达和纯化了具有功能的小鼠SCD1，并将其晶体结构解析到2.6？分辨率。 我们还表达和纯化了全长b5和b5R，并在体外组装了酶反应。在……里面 此外，我们还在b5R和b5之间以及b5和scd1之间生成了稳定的二元络合物，并且我们 建立了测定结合配对之间电子转移的分析方法。这些初步结果 使我们能够提出以下三个具体目标： 目的1：鉴定SCD1的结构和功能。 目的2：了解SCD1diIron中心的氧化和还原途径。 目的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.