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。血红素分子存在于血红蛋白中，是一种中心含有一个铁原子的卟啉。该项目将涉及生产血红素的钴类似物。这种钴-原卟啉（CoPPIX）辅因子预计将驱动以前在生物系统中未观察到的反应。 该项目还将支持一个新生本科研究经验课程的发展，重点是实验设计和分析。 塑料的酶分解将是结合教学/实验室课程中研究的模型系统。 将传统的合成催化剂与生物支架相结合是生物催化领域的一个重大挑战。由此产生的酶可以假设完成已知反应的高度选择性版本，甚至能够实现新的转化。交换金属辅因子的身份是产生新反应性的高效途径。然而，产生人工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。这项工作将试图开发一个强大的，可推广的路线，在体内生产钴取代卟啉（CoPPIX）。一旦完成，人们将探索如何控制这种辅因子的化学，以促进新的自然氢原子转移化学。本实验室已经证明E. coliBL 21可以合成CoPPIX并将其插入到蛋白质中。提出了三项相辅相成的任务：通过亚铁螯合酶工程改进CoPPIX生产，探索推定的钴-氢化物（Co-H）中间体的反应性，并调整携带CoPPIX辅因子的酶的化学选择性，以使用定向分子进化进行新的生物催化转化。该项目由ENG/该奖项反映了NSF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。