CAREER: Building and controlling the reactivity of a cobalt-porphyrin cofactor

酶是驱动生物反应的蛋白质。大多数工作都是与小分子合作进行的，这些小分子提供了反应所需的电子功能或分子片段。这些小分子，被称为辅因子，通常在其结构中包含一个或多个金属原子。这些金属通常是铁、锌、铜、镁和锰。一类重要的辅因子叫做卟啉。血红蛋白中的血红素分子是一种卟啉，其中心含有一个铁原子。该项目将涉及生产血红素的钴类似物。这种钴-原卟啉（CoPPIX）辅因子有望驱动以前未在生物系统中观察到的反应。该项目还将支持大一本科生研究经验课程的发展，重点是实验设计和分析。塑料的酶解将成为本课程的模型系统。将传统的合成催化剂与生物支架相结合是生物催化的一大挑战。由此产生的酶可以完成已知反应的高选择性版本，甚至可以实现新的转化。交换金属辅因子的身份是产生新反应性的高效途径。然而，合成人工金属酶的过程是费力的。该项目的中心目标是利用一种新的、完全遗传编码的金属辅助因子来扩大生物化学的反应范围。这项工作将试图开发一种强大的，可推广的途径来生产钴取代卟啉（CoPPIX）在体内。一旦完成，人们将探索如何控制这种辅助因子的化学性质，以促进新的自然氢原子转移化学。该实验室已经证明大肠杆菌BL21可以被用来生物合成CoPPIX并将该辅因子插入蛋白质中。他们提出了三个互补的任务：通过铁chetalase工程提高CoPPIX的产量，探索一种假定的钴氢化物（Co-H）中间体的反应性，以及利用定向分子进化调整含有CoPPIX辅助因子的酶的化学选择性，以进行新的生物催化转化。该项目由英国工程技术学院/中国生物工程学院的细胞和生化工程（CBE）项目以及MPS/CHE的生命过程化学（CLP）和化学催化（CAT）项目共同支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Enzymes are proteins that drive biological reactions. Most work in concert with small molecules that contribute electronic functions or molecular pieces needed for the reaction. These small molecules, referred to as cofactors, often contain one or more metal atoms in their structure. These metals are most often iron, zinc, copper, magnesium, and manganese. An important class of cofactors are called porphyrins. The heme molecule, found in hemoglobin, is a porphyrin that contains an iron atom in its center. This project will involve the production of a cobalt analog of heme. This cobalt-protoporphyrin (CoPPIX) cofactor is expected to drive reactions that have not been previously observed to occur in biological systems. The project will also support the development of a freshman undergraduate research experience course focused on experimental design and analysis. The enzymatic breakdown of plastic will be the model system studied in the combined instruction/lab course. A grand challenge in biocatalysis is to merge traditional synthetic catalysts with biological scaffolds. The resulting enzymes could hypothetically accomplish highly selective versions of known reactions and even enable new transformations. Swapping the identity of a metal cofactor is a highly productive route to generating new reactivity. However, the process of generating artificial metalloenzymes is laborious. The central goal of this project is to expand the reaction scope of biochemistry using a new, fully genetically encoded metallo-cofactor. This effort will attempt to develop a robust, generalizable route to producing cobalt-substituted porphyrin (CoPPIX) in vivo. Once accomplished, one would explore how the chemistry of this cofactor can be controlled to facilitate new-to-nature hydrogen-atom transfer chemistry. This lab has already shown that E. coli BL21 can be coopted to biosynthesize CoPPIX and insert this cofactor into proteins. Three complementary tasks are proposed: improve CoPPIX production through ferrochetalase engineering, explore the reactivity of a putative cobalt-hydride (Co-H) intermediate, and tune the chemoselectivity of enzymes bearing the CoPPIX cofactor to perform new biocatalytic transformations using directed molecular evolution.This project is being jointly supported by the Cellular and Biochemical Engineering (CBE) Program in ENG/CBET and by the Chemistry of Life Processes (CLP) and Chemical Catalysis (CAT) Programs in MPS/CHE.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.