Expanding the Enzyme Repertoire by Evolution and Engineering

创造新的酶（加速化学反应的蛋白质）是一项艰巨的挑战，目前几乎不存在有效的设计策略。然而，大自然经常创造新的酶，例如当需要酶来降解人造化学物质并将其用作营养源时。该项目将基于对化学和酶自然进化的基本理解，制定创造新酶的策略。该项目将探讨天然存在的酶的范围可以在多大程度上扩展以提供新颖的化学物质，并且具有为化学和制药行业的竞争力做出贡献的巨大潜力。该项目将提供跨学科培训和准备，帮助研究生和博士后在快速变化的学术和专业环境中发展职业。该项目扩展了概念验证，证明由于酶的多功能铁血红素辅基，细胞色素 P450 的催化功能可以将合成前体适应新的反应中间体。这些新的反应中间体开辟了一系列自然界未知的酶催化反应的可能性。该项目将探索如何充分优化非自然反应动力学，以及如何结合体内和体外方法将新反应纳入代谢途径。定向进化将用于改善突变细胞色素 P450 的表达，改善反应动力学，并扰乱电子转移速率以提高氮宾转移反应的选择性。对所有进化中间体的“化石记录”进行详细的动力学、结构和生化研究将为实现新的胺化活性的分子途径提供基本的见解。对分子间烯烃氮丙啶化和 C-H 胺化的特别关注利用了定向进化方面的专业知识以及对 P450 天然化学和所需新酶活性之间机制相似性的理解。该奖项由分子和细胞生物科学部系统和合成生物学计划联合资助；生命过程化学项目，化学部，以及化学、生物工程、环境和运输系统部的生物催化项目。

Creating new enzymes (proteins that speed up chemical reactions) is a formidable challenge for which few effective design strategies exist. However, nature frequently creates new enzymes, for example when enzymes are needed to degrade man-made chemicals and to use these as a nutrient source. This project will develop a strategy for creating new enzymes based on a fundamental understanding of both chemistry and of the natural evolution of enzymes. The project will probe the degree to which the range of naturally occurring enzymes can be expanded to deliver novel chemistries and has significant potential to contribute to the competitiveness of the chemical and pharmaceutical industries. The project will provide interdisciplinary training and preparation that will help graduate students and postdoctoral fellows to develop careers in a rapidly changing academic and professional environment. The project extends a proof-of-concept demonstration that the catalytic functions of cytochrome P450s can accommodate synthetic precursors to new reactive intermediates as a result of the enzymes' versatile iron-heme prosthetic group. These new reactive intermediates open up the possibility of an array of enzyme-catalyzed reactions that are not known in nature. The project will explore how the non-natural reaction kinetics can be fully optimized and how the new reactions can be incorporated into metabolic pathways using a combination of in vivo and in vitro approaches. Directed evolution will be used to improve the expression of mutated cytochrome P450s, improve reaction kinetics, and perturb the rate of electron transfer for improved nitrene-transfer reaction selectivity. Detailed kinetic, structural and biochemical studies of the 'fossil record' of all evolutionary intermediates will provide fundamental insights into the molecular pathways by which new amination activities can be realized. The specific focus on intermolecular alkene aziridination and C-H amination draws on expertise in directed evolution and understanding of mechanistic similarities between P450's native chemistry and the desired new enzyme activities.This award is funded jointly by the Systems and Synthetic Biology Program, Division of Molecular and Cellular Biosciences; the Chemistry of Life Processes Program, Division of Chemistry, and the Biocatalysis Program of the Chemical, Bioengineering, Environmental, and Transport Systems Division.