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，几乎没有有效的设计策略。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品并将其用作营养源时。该项目将根据对化学和酶的自然进化的基本理解，开发一种创造新酶的策略。该项目将探索天然酶的范围可以扩大到何种程度，以提供新的化学物质，并具有显著的潜力，以促进化学和制药行业的竞争力。该项目将提供跨学科培训和准备，帮助研究生和博士后研究员在快速变化的学术和专业环境中发展职业生涯。该项目扩展了概念验证的演示，即细胞色素P450的催化功能可以将合成前体容纳为新的活性中间体，这是由于酶的多功能铁血红素辅基。这些新的活性中间体开辟了一系列自然界中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天然反应动力学，以及如何使用体内和体外方法的组合将新反应纳入代谢途径。定向进化将用于改善突变的细胞色素P450的表达，改善反应动力学，并扰动电子转移速率以改善氮转移反应的选择性。详细的动力学，结构和生物化学研究的“化石记录”的所有进化中间体将提供基本的见解的分子途径，新的胺化活动可以实现。该奖项特别关注分子间烯烃氮丙啶化和C-H胺化，利用了定向进化和理解P450天然化学和所需新酶活性之间的机制相似性的专业知识。生命过程的化学程序，化学部，和化学，生物工程，环境和运输系统部的生物催化程序。

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.