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

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.