Enzymes with an Expanded Amino Acid Alphabet

结合计算设计和定向进化是为非生物反应创造酶的有效方法。然而，可获得的化学物质的范围受到自然正则氨基酸字母表的限制，这些正则氨基酸含有有限的功能，并且对于许多生物催化转化并不是最佳的。遗传密码扩展(GCE)允许在酶活性位点上选择性地安装“化学编程”的非正则氨基酸(NCAA)，从而提供了访问自然界中没有的有机催化机制的机会。一个有用的例子是硫脲，这是一类有价值的小分子有机催化剂，能够通过双重氢键作用激活羰基化合物，促进广泛的化学转化。在该组之前，已成功地设计了吡咯酰-tRNA合成酶/PyltRNACUA对，以编码几个赖氨酸衍生的硫脲，以响应重新分配的琥珀终止密码子(UAG)。我们目前正在努力扩大可编码硫脲的范围，到目前为止已经成功地纳入了一种尿素类似物。有了这些翻译组件，我们将能够探索通过计算设计的活性位点中的一系列化学转换，最终通过定向进化优化这些酶。因此，这种方法结合了蛋白质催化剂的效率和选择性，以及小分子有机催化剂可获得的广泛反应范围。

Combining computational design and directed evolution is a powerful approach to create enzymes for non-biological reactions. However, the range of chemistries accessible is restricted by Nature's alphabet of canonical amino acids, which contain limited functionality and are not optimal for many biocatalytic transformations. Genetic code expansion (GCE) allows the site-selective installation of 'chemically programmed' non-canonical amino acids (ncAAs) into enzyme active sites, providing opportunities to access organocatalytic mechanisms not represented in Nature. A useful example are thioureas, which are a valuable class of small-molecule organocatalysts capable of activating carbonyl compounds through dual hydrogen-bonding interactions to promote a broad range of chemical transformations.Previously in the group, pyrrolysyl-tRNA synthetase/PyltRNACUA pairs have successfully been engineered to encode several lysine-derived thioureas in response to reassigned amber stop codons (UAG). We are currently working on expanding the scope of encodable thioureas and have so far successfully incorporated one urea analogue. With these translation components in hand, we will be able to explore a range of chemical transformations within computationally designed active sites, eventually optimising these enzymes via directed evolution. This approach thus combines the efficiency and selectivity of protein catalysts, with the broad range of reactivities accessible with small molecule organocatalysts.