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，然后必须测试这些化合物的相关和强大的功能。酶是自然界强大的化学家，简单而安全地执行许多具有人工挑战性的化学转化。该基金的目的是利用高通量技术来快速测试酶的功能和底物的混杂程度，以实现能够取代危险和昂贵的合成化学方法的酶。利用“mRNA显示”，这是一种产生和筛选大量核苷酸编码多肽库的技术，将合成数百万潜在的酶底物，并对其进行酶修饰测试。这一简化的过程将提供对酶混杂的快速、全面的分析，以揭示酶在有机合成应用中的用处。该项目将在日本东京大学信使核糖核酸显示专家菅义明博士的指导下进行。Lynd是一种假定的杂交酶，作用于核糖体合成和翻译后修饰的多肽(RIPP)天然产物的多肽底物。Lynd的目的是将半胱氨酸残基转化为噻唑啉--这通常是RIPP生物活性所必需的转化。利用mRNA展示技术，将合成数百万种独特的Lynd底物，并将其附着在遗传条形码上。被Lynd完全修饰的底物将使用与未修饰的半胱氨酸发生共价反应的树脂进行分离。一旦修饰的底物被提纯，遗传条形码将被测序，以揭示Lynd的全面底物混杂。如果在修饰和未修饰的酶底物之间提供适当的选择技术，可以预见本奖项中使用的方法将扩展到其他多肽修饰酶。该奖项根据东亚和太平洋夏季学院计划，支持一名美国研究生的暑期研究，由NSF和日本科学促进会共同资助。

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.