Design and Evolution of Enzymes with Non-Canonical Amino Acids

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，它们的催化机制通常由氨基酸侧链上的化学基团组成。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想象的机制范围。这就提出了一个有趣的问题：酶的催化功能是否可以通过使用一个扩展的氨基酸“字母表”来扩展，从而提供更广泛的催化侧链。近年来，我们的团队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.）。化学。社会科学学报，2018,35(4):755 - 755。化学。Soc. 2016, 138, 11344)。我们的方法利用工程细胞翻译组件来选择性地安装含有功能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，基因编码非规范功能提供了巨大的优势：它极大地促进了定义良好的、均匀的蛋白质的产生；它允许非规范氨基酸被引入到任何位置，任何蛋白质支架中；而且，也许最重要的是，它允许使用定向进化快速优化酶的性质。受小分子有机催化机制策略的启发，我们最近结合了遗传密码扩展、计算酶设计和实验室进化，创造了利用非规范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，这将在博士学位期间得到充分的探索。不受遗传密码的限制，学生将使用我们先进的酶工程技术来创造具有在自然界中未观察到的功能的酶，这些酶以前被认为是生物催化领域无法实现的。该项目将特别致力于创建含有嵌入在所设计的活性位点内的功能性n -杂环碳烯（NHC）基序的酶。近年来，NHCs作为有机催化剂和过渡金属（如钌、金、钯）催化剂（包括第二代复分解催化剂）的配位体，在合成化学中成为功能强大且用途广泛的官能团（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催化剂提供巨大的机会，以促进大量重要的化学转化。为了实现这一目标，我们将利用实验室现有的工程细胞翻译成分，将NHCs作为辅助因子嵌入设计的活性位点，并随后探索这些辅助因子作为有机催化剂和作为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，提高催化效率并调整NHC辅因子的电子和结构性质。值得注意的是，有希望的初始设计可以通过迭代轮次的定向进化来大幅改进，以提供高效和选择性的新酶来生产高价值分子。该项目采用真正创新的方法，融合了生物催化、有机催化和过渡金属催化领域，因此与iCAT网络的战略重点完全一致。

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.