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

职业：构建和控制钴卟啉辅因子的反应性

• 批准号: 2237213
• 负责人: Andrew Buller
• 金额: $ 70.88万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2237213&HistoricalAwards=false
• 关键词: CAREER; Building; controlling; reactivity; cobalt

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.

酶是驱动生物反应的蛋白质。大多数与小分子协同工作，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，它们的结构中通常含有一个或多个金属原子。这些金属通常是铁、锌、铜、镁和锰。一类重要的辅因子被称为卟啉。血红素分子是一种中心含有铁原子的卟啉，存在于血红蛋白中。该项目将涉及生产一种类似于亚铁血红素的钴。这种钴-原卟啉(CoPPIX)辅助因子有望推动以前在生物体系中未曾观察到的反应。该项目还将支持开发一门以实验设计和分析为重点的一年级本科生研究体验课程。塑料的酶分解将是教学/实验相结合课程中研究的模型系统。生物催化领域的一个重大挑战是将传统的合成催化剂与生物支架相结合。由此产生的酶可以假设完成已知反应的高度选择性版本，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，产生人造金属酶的过程是费力的。该项目的中心目标是使用一种新的、完全遗传编码的金属辅因子来扩大生物化学的反应范围。这一努力将试图开发一种在体内生产钴取代卟啉(CoPPIX)的可靠、可推广的方法。一旦完成，人们将探索如何控制这种辅因子的化学，以促进新的性质的氢原子转移化学。这个实验室已经证明，大肠杆菌BL21可以被增选来生物合成CoPPIX，并将这种辅因子插入蛋白质中。提出了三项相辅相成的任务：通过FeChetalase工程提高CoPPIX的产量，探索假定的钴-氢化物(Co-H)中间体的反应活性，以及调整携带CoPPIX辅因子的酶的化学选择性，以使用定向分子进化执行新的生物催化转化。该项目由ENG/CBET中的细胞和生化工程(CBE)计划以及MPS/CHE中的生命过程化学(CLP)和化学催化(CAT)计划联合支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，认为值得支持。