Exploiting the distinctive catalysis of chemically modified enzymes

利用化学修饰酶的独特催化作用

• 批准号: BB/N002091/1
• 负责人: Alan Berry
• 金额: $ 61.1万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN002091%2F1
• 关键词: Exploiting; distinctive; catalysis; chemically; modified

Enzymes are the catalysts that nature uses to accelerate chemical reactions. Because enzymes are spectacularly efficient catalysts that operate in water at room temperature, they have tremendous potential to be exploited in the 'green' manufacture of chemicals such as drug molecules. However, a major limitation for application in chemical synthesis is that enzymes are highly selective for a particular starting material, and it is not always possible to find an enzyme that nature has evolved that is suitable for a specific application. We propose to modify a specific enzyme such that it may be exploited in the catalysis of a much wider range of useful chemical transformations.Enzymes are proteins that are constructed from 20 amino acid building blocks. Essentially, the sequence of amino acids in a protein is analogous to a sequence of coloured beads in a necklace. Although nature exploits some very useful amino acid building blocks, the functions of enzymes are nonetheless limited by the fact that there are only 20 amino acids that are usually used to construct proteins. Here, we propose to exploit new methods to prepare proteins with a much wider range of amino acid building blocks. We have demonstrated the ability to position these new amino acids, termed non-canonical amino acids, throughout the active site of an enzyme and that the specificity of the enzyme may be changed by using the non-canonical amino acids in ways that cannot be achieved using the 'natural' building blocks. In this proposal we will first expand the range of specificity of the enzyme by positioning the new amino acids at a range of positions and then using an established assay we will find new reactions that can occur only when the new amino acid is present. In the second part of the work we will develop new assays to allow us to extend the possibilities even further. Firstly we will make use of nuclear magnetic resonance (NMR) to follow the reaction using labelled substrates, and then we will make use of NMR techniques from the realm of drug discovery (named fragment-based drug design) to search for a wider range of substrates used when we have non-canonical amino acids in the enzyme. We will also extend the chemical method for incorporation of the non-canonical amino acid.Finally, and most excitingly, the ability to insert 'unnatural' amino acid building blocks also opens the way to building completely novel enzyme chemistries - possible using one of the inserted 'unnatural' amino acids, but not possible using any of the 20 natural amino acids. For this we have taken inspiration from a field of chemistry termed organocatalysis. Here relatively simple organic molecules can catalyse reactions, and the coupling of this area with the specificity and catalytic power of enzymes will be used to generate enzymes with huge potential for new, greener routes to a range of important chemicals.The modified enzymes that we will discover will be able to catalyse reactions for which enzymes are not currently available. Such enzymes would have tremendous value in the 'green' synthesis of complex biologically active molecules such as drugs.

酶是自然界用来加速化学反应的催化剂。由于酶是非常有效的催化剂，可以在室温下在水中运行，因此它们在药物分子等化学品的“绿色”制造中具有巨大的潜力。然而，在化学合成中应用的一个主要限制是，酶对特定的起始物质具有高度的选择性，并且并不总是可能找到一种自然进化的适合特定应用的酶。我们建议对一种特定的酶进行修饰，使其可以用于催化更广泛的有用的化学转化。酶是由20个氨基酸组成的蛋白质。从本质上讲，蛋白质中的氨基酸序列类似于项链上的一串彩色珠子。尽管自然界利用了一些非常有用的氨基酸组成成分，但酶的功能仍然受到这样一个事实的限制，即通常只有20个氨基酸用于构建蛋白质。在这里，我们建议开发新的方法来制备具有更广泛的氨基酸组成单元的蛋白质。我们已经证明了这些新的氨基酸，称为非规范氨基酸，在酶的活性部位定位的能力，并且使用非规范氨基酸可以改变酶的特异性，而使用‘天然’构建块无法实现。在这项建议中，我们将首先通过将新氨基酸定位在一系列位置来扩大酶的特异性范围，然后使用已建立的分析方法，我们将发现只有当新氨基酸存在时才能发生的新反应。在这项工作的第二部分，我们将开发新的分析方法，使我们能够进一步扩大可能性。首先，我们将利用核磁共振(NMR)跟踪标记底物的反应，然后我们将利用药物发现领域的核磁共振技术(称为基于片段的药物设计)来寻找当酶中含有非规范氨基酸时使用的更广泛的底物。我们还将扩展非规范氨基酸的化学方法。最后，最令人兴奋的是，插入“非天然”氨基酸构件的能力也为构建全新的酶化学开辟了道路--可能使用插入的“非天然”氨基酸中的一种，但不可能使用20种天然氨基酸中的任何一种。为此，我们从一个被称为有机催化的化学领域获得了灵感。在这里，相对简单的有机分子可以催化反应，这一区域与酶的特异性和催化能力的结合将被用来产生具有巨大潜力的酶，为一系列重要的化学物质提供新的、更绿色的路线。我们将发现的修饰酶将能够催化目前酶不存在的反应。这种酶在药物等复杂生物活性分子的“绿色”合成方面将具有巨大的价值。

项目成果

Extending enzyme molecular recognition with an expanded amino acid alphabet.

通过扩展的氨基酸字母表扩展酶分子识别。

• DOI: 10.1073/pnas.1616816114
• 发表时间: 2017
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Windle CL

Aldolase-catalysed stereoselective synthesis of fluorinated small molecules.

醛缩酶催化氟化小分子的立体选择性合成。

• DOI: 10.1016/j.cbpa.2016.12.029
• 发表时间: 2017
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8
• 作者: Windle CL