Metalloenzyme structure, function and assembly

摘要 金属离子与蛋白质的结合提供了独特的化学反应，这是许多 自然界最重要和最令人惊叹的化学变化。例如，金属酶催化 将核糖核苷酸还原为脱氧核糖核苷酸，这是DNA生物合成中的一个限速步骤。他们 生物合成具有特殊支架的抗癌和抗病毒化合物并催化碳-碳 在二氧化碳和氢气上提供生命的成键反应。我们的实验室采用结构方法来审问 金属酶是如何完成这种不可思议的化学反应的。我们试图理解 金属酶的结构允许自由基物种被控制--即关闭、开启和 驾驭--以实现手边的反应。我们还努力了解蛋白质是如何被设计成能够 长程电子转移，无蛋白质损伤或自由基损失。在本提案中，我们描述了结构 我们的金属酶模型体系的研究，包括Ia类(依赖二铁)和III类(甘氨酸根- 依赖的)核糖核苷酸还原酶，使我们能够询问基于自由基的分子基础 化学反应。我们还描述了理解蛋白质支架如何促进有机金属化学的努力， 特别是在微生物固定二氧化碳和产生甲烷方面。这些研究充分利用了 我们在氧气敏感金属酶和低温电子显微镜(Cryo-EM)方面的专业知识。 尽管我们将继续使用X射线结晶学，但低温EM被证明是许多人的游戏规则改变者 我们的金属酶系统。特别是，低温电磁的分辨率革命为我们提供了 获得期待已久的大型(2000 KDa)和瞬时金属蛋白络合物的结构，并 确定金属酶在功能必需的构象状态下的结构 结晶学无法达到的。我们的结构研究结果将使基于结构的设计成为可能 以微生物核糖核苷酸还原酶为靶点的新型抗生素。这些结构性数据还将 指导开发自由基酶以生产具有特殊医学意义的化合物的努力 脚手架。这些数据将进一步促进金属酶或其仿生无机材料的应用。 从温室气体二氧化碳生产高价值碳化合物的同行，理想情况下， 提高我们对金属蛋白生物化学中一些更神秘的方面的理解。

Abstract The combination of metal ions with proteins offers unique chemical reactivities that are at the heart of many of Nature’s most important and amazing chemical transformations. For example, metalloenzymes catalyze the reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA biosynthesis. They biosynthesize anticancer and antiviral compounds that have unusual scaffolds and catalyze the carbon-carbon bond forming reactions that afford life on CO2 and H2 gases. Our lab employs structural methods to interrogate how metalloenzymes are able to perform this incredible chemistry. We seek to understand how the architecture of metalloenzymes allows for radical species to be controlled – i.e. turned off, turned on and harnessed – to enable the reaction at hand. We also strive to understand how proteins are designed to enable long-range electron transfer without protein damage or radical loss. In this proposal, we describe structural studies of our metalloenzyme model systems, including class Ia (diiron-dependent) and class III (glycyl radical- dependent) ribonucleotide reductases that allow us to interrogate the molecular basis of radical-based chemistry. We also describe efforts to understand how protein scaffolds facilitate organometallic chemistry, especially in regard to microbial carbon dioxide fixation and methane production. These studies leverage both our expertise in working with O2-sensitive metalloenzymes and in cryogenic-electron microscopy (cryo-EM). Although we will continue to employ X-ray crystallography, cryo-EM is proving to be a game-changer for many of our metalloenzyme systems. In particular, the resolution revolution of cryo-EM provides us with the means to obtain long-awaited structures of both large (2000 kDa) and transient metalloprotein complexes and to determine structures of metalloenzymes in functionally-essential conformational states that were previously unattainable by crystallography. The results of our structural studies will enable structure-based design of novel antibiotics targeting, for example, microbial ribonucleotide reductases. These structural data will also guide efforts to exploit radical enzymes for the production of medically important compounds with unusual scaffolds. These data will additionally facilitate the application of metalloenzymes or their bioinspired inorganic counterparts in the production of high-value carbon compounds from the greenhouse gas CO2 and, ideally, improve our understanding of some of the more enigmatic aspects of metalloprotein biochemistry.