2023 103 P

Composing Point Mutations of E33 in the VcSpeG Enzyme to Study Allosteric Effector Molecule Recognition and Effects on Enzymatic Activity

By: Tiffany Chambers

Department: Chemistry & Biochemistry

Faculty Advisor: Dr. Misty Kuhn

The occurrence of mutations in proteins can be used to gain insight into how an allosteric signal is transmitted and also how amino acids change enzymatic activity. Our laboratory studies Gcn5-related N-acetyltransferases (GNATs), which are significant for this research because they are involved in numerous biological processes. This family of enzymes is responsible for acylating proteins, peptides, antibiotics, polyamines, and other molecules. One specific GNAT that is the focus of this research project is the Vibrio cholerae SpeG (VcSpeG) spermidine N-acetyltransferase from the bacterium Escherichia-coli. This protein acetylates spermidine and spermine which are polyamines. Polyamines are important in bacteria because they are needed for their physiological functions like making biofilms, growing, making natural products, and surface behaviors. They are also important for binding nucleotides. There is currently not much known about how to residues of the VcSpeG enzyme allosteric site transmit an allosteric signal and affect enzyme activity. One residue that is located in the allosteric site is E33, which forms H-bonds to polyamines in crystal structures of the VcSpeG enzyme. We wanted to investigate the impact of E33 on VcSpeG enzymatic activity, and how the allosteric signal is transmitted. Therefore, we first generated a series of point mutations of the E33 residue and then purified these proteins using Ni2+-affinity chromatography. Here we present our progress on this project, which will ultimately help to clarify the role of this residue.