Substrate channelling in catabolism of methylated amines

甲基化胺分解代谢中的底物通道

• 批准号: BB/E017010/1
• 负责人: David Leys
• 金额: $ 86.87万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE017010%2F1
• 关键词: Substrate; channelling; catabolism; methylated; amines

The many chemical reactions supporting life are catalysed by enzymes. Some of the reactions that occur naturally can give rise to unstable compounds. If left unattented by the cellular machinery, this can be toxic to the host organism. To avoid this, many enzymes guide these unstable compounds to the next enzyme in a process called substrate channelling. This involves the careful and intricate design of enzymes at the molecular level where the unstable compound is transferred through narrow channels to its final destination. These channels ensure the compound does not escape into the bulk solution and thus does not undergo unwanted side reactions. How these enzyme systems have evolved is uncertain, although it has been proposed that the first 'channelling enzymes' were far from perfect, leaking unstable compounds at acceptable levels. We have recently elucidated the molecular detail of an enzyme (DMGO) that apparently mimics one of these early designs of a substrate channelling enzyme. IThe structure of DMGO is far simpler than other known channelling systems, yet it is widespread among organisms including humans, suggesting further significant improvement does not provide additional benefit to the organism or has not been possible to achieve. The protein in question catalyses the oxidation of methylated-amines. Enzymic oxidation of the methyl group can, over time, give rise to the release of toxic formaldehyde. To avoid this, the enzyme channels the oxidized product to a second active site where it transfers the oxidized methyl-group to tetrahydrofolate, an essential co-enzyme that acts as a resoervoir of one-carbon units. We will investigate if these simple enzymes leak unstable intermediate compounds as their simple design would suggest. Furthermore, we aim to gain atomic level insight into function of the molecular architecture by determining the structure of other members of this protein family. Using sophisticated structural biology techniques we aim to visualise what happens at active site 2 (the folate site) during transfer of oxidized-methyl groups. We will also focus our efforts on characterizing other proteins containing some key elements present in the bifunctional amine oxidases. We thus wish to determine whether this particular simple substrate channelling mechanism is widespread and has evolved in several distinct systems. Information gained from our studies will provide insight into the origins and mechanisms of substrate channelling, and provide a detailed understanding of channelling that could guide the next level of protein design and engineering through the incorporation of retaining nano-sized reservoirs in protein catalysts.

维持生命的许多化学反应都是由酶催化的。一些自然发生的反应可能会产生不稳定的化合物。如果不受细胞机制的影响，这可能对宿主生物体有毒。为了避免这种情况，许多酶在一个称为底物通道的过程中将这些不稳定的化合物引导到下一个酶。这涉及到酶在分子水平上的仔细和复杂的设计，其中不稳定的化合物通过狭窄的通道转移到其最终目的地。这些通道确保化合物不会逃逸到本体溶液中，因此不会发生不必要的副反应。这些酶系统是如何进化的尚不确定，尽管有人提出第一种“通道酶”远非完美，在可接受的水平上泄漏不稳定的化合物。我们最近阐明了一种酶（DMGO）的分子细节，这种酶显然模仿了底物通道酶的早期设计之一。DMGO的结构比其他已知的通道系统简单得多，但它在包括人类在内的生物体中广泛存在，这表明进一步的显著改进不会为生物体提供额外的益处或不可能实现。所讨论的蛋白质催化甲基化胺的氧化。随着时间的推移，甲基的酶促氧化会导致有毒甲醛的释放。为了避免这种情况，酶将氧化产物引导到第二个活性位点，在那里它将氧化的甲基转移到四氢叶酸，四氢叶酸是一种必需的辅酶，作为一个碳单元的resoervoir。我们将研究这些简单的酶是否会像它们简单的设计所暗示的那样泄漏不稳定的中间化合物。此外，我们的目标是通过确定该蛋白质家族其他成员的结构来获得原子水平的分子结构功能。使用复杂的结构生物学技术，我们的目标是可视化在氧化甲基转移过程中活性位点2（叶酸位点）发生了什么。我们还将集中精力对双功能胺氧化酶中含有一些关键元素的其他蛋白质进行表征。因此，我们希望确定这种特殊的简单的基板通道机制是否是广泛的，并已在几个不同的系统。从我们的研究中获得的信息将提供对底物通道的起源和机制的深入了解，并提供对通道的详细理解，从而可以通过在蛋白质催化剂中加入保留纳米尺寸的水库来指导下一阶段的蛋白质设计和工程。

项目成果

An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme

• DOI: 10.1038/nature20159
• 发表时间: 2016-11
• 期刊: Nature
• 影响因子: 64.8
• 作者: M. Ortmayer;P. Lafite;B. Menon;T. Tralau;K. Fisher;L. Denkhaus;N. Scrutton;S. Rigby;A. Munro;Sam Hay;D. Leys