Substrate channelling in catabolism of methylated amines

许多维持生命的化学反应都是由酶催化的。有些自然发生的反应会产生不稳定的化合物。如果不被细胞机制注意，这可能对宿主生物有毒。为了避免这种情况，许多酶在一个被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。这需要在分子水平上对酶进行细致而复杂的设计，使不稳定的化合物通过狭窄的通道转移到最终目的地。这些通道确保化合物不会逃逸到散装溶液中，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，第一个“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，它显然模仿了这些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给生物体带来额外的好处，或者不可能实现。所讨论的蛋白质催化甲基化胺的氧化。随着时间的推移，甲基的酶氧化会释放出有毒的甲醛。为了避免这种情况，酶将氧化产物引导到第二个活性位点，在那里它将氧化甲基转移到四氢叶酸，四氢叶酸是一种必不可少的辅酶，作为一碳单元的储存库。我们将研究这些简单的酶是否会泄漏不稳定的中间化合物，就像它们简单的设计所暗示的那样。此外，我们的目标是通过确定该蛋白家族其他成员的结构来获得原子水平上对分子结构功能的了解。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。我们还将集中精力表征含有双功能胺氧化酶中存在的一些关键元素的其他蛋白质。因此，我们希望确定这种特殊的简单基质通道机制是否广泛存在，并在几个不同的系统中进化。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，这可以指导蛋白质设计和工程的下一阶段，通过在蛋白质催化剂中加入保留纳米级储层。

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.