EAPSI: Using High-throughput Technologies to Quickly and Comprehensively Probe Enzymatic Function and Promiscuity

化学合成是开发用于生物材料、药物和化学探针的新分子的主要途径。化学家在合成这些化合物时经常使用危险，耗时和昂贵的技术，然后必须测试相关和强大的功能。酶是自然界强大的化学家，简单而安全地进行许多具有挑战性的合成化学转化。该资助的目的是利用高通量技术快速测试酶功能和底物混杂性，以实现能够取代危险和昂贵的合成化学方法的酶。使用“mRNA展示”，这是一种生成和筛选大量核苷酸编码肽库的技术，将合成数百万种潜在的酶底物并进行酶修饰测试。这个简化的过程将提供一个快速，全面的分析酶混杂揭示有用的酶的有机合成应用。该项目将在日本东京大学mRNA展示专家Hiroaki Suga博士的指导下进行。LynD是一种在核糖体合成和后修饰肽（RiPP）天然产物的成熟过程中作用于肽底物的puronically promiscuous酶。LynD的目的是将半胱氨酸残基转化为噻唑啉-通常是RiPP生物活性所必需的转化。使用mRNA展示技术，LynD的数百万个独特底物将被合成并连接到遗传条形码上。将使用与未修饰的半胱氨酸共价反应的树脂分离由LynD完全修饰的底物。一旦修饰的底物被纯化，将对遗传条形码进行测序以揭示LynD的全面底物混杂性。考虑到在修饰的和未修饰的酶底物之间的适当选择技术，可以设想，在该奖项中使用的方法将扩展到其他肽修饰酶。 该奖项是东亚和太平洋夏季研究所计划下的一个奖项，由美国国家科学基金会和日本科学促进会共同资助，支持美国研究生的夏季研究。

Chemical synthesis is the main route to develop new molecules for use in biomaterials, pharmaceutical drugs, and as chemical probes. Chemists often use dangerous, time-consuming, and costly techniques when synthesizing these compounds which must then be tested for relevant and robust function. Enzymes are nature's powerful chemists, performing many synthetically challenging chemical transformations simply and safely. It is the purpose of the grant to utilize a high-throughput technique to rapidly test enzymatic function and substrate promiscuity to realize enzymes able to replace dangerous and expensive synthetic chemistry methods. Using "mRNA display", which is a technology to generate and screen massive nucleotide-encoded peptide libraries, millions of potential enzyme substrates will be synthesized and tested for enzymatic modification. This streamlined process will provide a quick, comprehensive analysis of enzyme promiscuity to reveal the usefulness of an enzyme for organic synthesis applications. The project will be conducted under the guidance of Dr. Hiroaki Suga, an expert on mRNA display, at the University of Tokyo in Japan. LynD is a putatively promiscuous enzyme that acts on peptide substrates in the maturation of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. It is the purpose of LynD to transform cysteine residues into thiazolines -- often a necessary transformation for RiPP biological activity. Using mRNA display techniques, millions of unique substrates for LynD will be synthesized and attached to genetic barcodes. Substrates that are fully modified by LynD will be isolated using a resin that covalently reacts with unmodified cysteines. Once modified substrates are purified, the genetic barcodes will be sequenced to reveal the comprehensive substrate promiscuity of LynD. Given a proper selection technique between modified and unmodified enzyme substrates, it is envisioned that the processes used in this award will be expandable to other peptide modifying enzymes. This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the Japan Society for the Promotion of Science.