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Design and Evolution of Enzymes with Non-Canonical Amino Acids

非规范氨基酸酶的设计和进化

基本信息

批准号：
2465805
负责人：
金额：
--
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=studentship-2465805
关键词：
Design Evolution Enzymes Non Canonical

项目摘要

Enzymes are exceptionally powerful catalysts that recognize molecular substrates and process them in active sites. They are generally built from just 20 amino acids, and their catalytic machinery is typically assembled from chemical groups in the amino-acid side chains. But fewer than half of these side chains contain functional groups that can participate in enzyme catalytic cycles, which severely restricts the range of mechanisms conceivable within enzyme active sites. This raises the intriguing question of whether the catalytic repertoire of enzymes could be expanded by using an extended 'alphabet' of amino acids that offers a wider range of side chains for catalysis. In recent years our group have begun to take major strides towards achieving this ambitious vision (e.g. Nature 2019, 570, 219, ACS Catalysis, 2020, 10, 2735, J. Am. Chem. Soc. 2018, 140, 1535, J. Am. Chem. Soc. 2016, 138, 11344).Our approach exploits engineered cellular translation components to selectively install non-canonical amino acids containing functional side chains. Genetically encoding the non-canonical functionality offers enormous advantages over alternative methods for chemically modifying protein structure: it greatly facilitates the production of well-defined, homogeneous proteins; it allows the non-canonical amino acid to be introduced at any site, in any protein scaffold; and, perhaps most significantly, it allows for rapid optimization of enzyme properties using directed evolution. Inspired by mechanistic strategies from small molecule organocatalysis, we have recently employed a combination of genetic code expansion, computational enzyme design and laboratory evolution to create enzymes that exploit non-canonical amino acids as catalytic nucleophiles (Nature 2019, 570, 219). This study now opens up new and exciting opportunities to enzyme designers and engineers which will be fully explored within this PhD studentship. Free from the constraints of the genetic code, the student will employ our advanced enzyme engineering techniques to create enzymes with functions not observed in Nature, that were previously thought inaccessible to the field of biocatalysis. The project will specifically aim to create enzymes that contain a functional N-heterocyclic carbene (NHC) motif embedded within the designed active site. In recent years, NHCs have emerged as powerful and highly versatile functional groups in synthetic chemistry, both as organic catalysts and as a coordinating ligands to transition metal (e.g. ruthenium, gold, palladium) catalysts including 2nd generation metathesis catalysts (Nature 2014, 510, 485). The development of general strategies for incorporating NHCs into protein active sites will therefore offer great opportunities to create highly efficient and selective catalysts for wealth of important chemical transformations. To address this objective, we will exploit engineered cellular translation components available in our laboratory to embed NHCs as cofactors into designed active sites, and subsequently explore the applications of these cofactors as organic catalysts and as ligands for gold and ruthenium mediated processes. Here we can take advantage of molecular recognition elements provided by the protein scaffold to achieve enantioselective conversions, to enhance catalytic efficiencies and to tune the electronic and structural properties of NHC cofactor. Significantly, promising starting designs can be substantially improved through iterative rounds of directed evolution to afford highly efficient and selective de novo enzymes for the production of high value molecules. The project takes a truly innovative approach to merge the fields of biocatalysis, organocatalysis and transition metal catalysis, and thus is perfectly aligned to the strategic priorities of the iCAT network.

酶是非常强大的催化剂，它能识别分子底物并在活性位点处理它们。它们通常由20个氨基酸组成，其催化机制通常由氨基酸侧链中的化学基团组装而成。但这些侧链中只有不到一半含有可以参与酶催化循环的官能团，这严重限制了酶活性位点内可能的机制范围。这就提出了一个有趣的问题，即是否可以通过使用扩展的氨基酸“字母表”来扩展酶的催化能力，从而提供更广泛的催化侧链。近年来，我们的团队已经开始朝着实现这一雄心勃勃的愿景迈出重大步伐（例如，Nature 2019，570，219，ACS Catalysis，2020，10，2735，J. Am. 2018，140，1535，J. Am. 2016，138，11344）〇我们的方法利用工程化的细胞翻译组分来选择性地安装含有功能性侧链的非规范氨基酸。与化学修饰蛋白质结构的替代方法相比，遗传编码非规范功能性提供了巨大的优势：它极大地促进了定义明确的同质蛋白质的生产;它允许在任何蛋白质支架中的任何位点引入非规范氨基酸;并且，也许最重要的是，它允许使用定向进化快速优化酶特性。受小分子有机催化的机械策略的启发，我们最近采用了遗传密码扩展，计算酶设计和实验室进化的组合来创建利用非典型氨基酸作为催化亲核试剂的酶（Nature 2019，570，219）。这项研究现在为酶设计师和工程师开辟了新的和令人兴奋的机会，这些机会将在这个博士生项目中得到充分的探索。不受遗传密码的限制，学生将采用我们先进的酶工程技术来创建具有自然界中未观察到的功能的酶，这些功能以前被认为是生物催化领域无法实现的。该项目将专门致力于创建包含嵌入设计活性位点内的功能性N-杂环卡宾（NHC）基序的酶。近年来，NHC已成为合成化学中强大且用途广泛的官能团，既可作为有机催化剂，也可作为过渡金属（例如钌、金、钯）催化剂（包括第二代复分解催化剂）的配位配体（Nature 2014，510，485）。因此，将NHCs纳入蛋白质活性位点的一般策略的发展将为创造高效和选择性的催化剂提供巨大的机会，用于丰富的重要化学转化。为了实现这一目标，我们将利用我们实验室提供的工程细胞翻译组件，将NHC作为辅因子嵌入设计的活性位点，随后探索这些辅因子作为有机催化剂和金和钌介导过程的配体的应用。在这里，我们可以利用蛋白质支架提供的分子识别元件来实现对映选择性转化，提高催化效率并调节NHC辅因子的电子和结构性质。值得注意的是，有希望的起始设计可以通过迭代轮的定向进化得到实质性改进，以提供用于生产高价值分子的高效和选择性的从头酶。该项目采用真正创新的方法，将生物催化、有机催化和过渡金属催化领域融合在一起，从而与iCAT网络的战略重点完美契合。